Identification and Characterization of reference genes

from Sorghum genome for qPCR based expression

analysis

Major Project Thesis

Submitted by

NIKITA GUPTA

For the partial fulfilment of the

Degree of Master of Science in
PLANT BIOTECHNOLOGY

Submitted to

Department of Biotechnology
TERI University
2016

DECLARATION

This is to certify that the work embodied in this thesis titled Identification and

Characterization of reference genes from Sorghum genome for qPCR based expression
analysis is an original work carried out by me and has not been submitted anywhere else for the
award of any degree.

I certify that all sources of information and data are fully acknowledged in the project thesis.

NIKITA GUPTA
Date: 16th May, 2016

CERTIFICATE

This is to certify that Ms.NIKITA GUPTA has carried out her major project in partial
fulfilment of the requirement for the degree of Master of Science in PLANT
BIOTECHNOLOGY on the topic Identification and Characterization of reference genes
from Sorghum genome for qPCR based expression analysis during December 2015 to May
2016. The project was carried out at the School of Biotechnology, JNU New Delhi.

To the best of our knowledge the thesis embodies the original work of the candidate.

Date: 16th May, 2016

Dr. Manoj K. SharmaDr. Sonika Gupta

(External Supervisor) (Internal Supervisor)
Assistant Professor

Assistant Professor

School of Biotechnology, JNU Department of Biotechnology

New Delhi-110067. TERI University, New Delhi.

Dr. Anandita Singh

Associate Professor & Head
Department of Biotechnology
TERI University
New Delhi

Acknowledgement
I would like express my deep gratitude to my supervisor, Dr. Manoj K. Sharma for allowing
me to carry out my dissertation with his research group in Plant Molecular Biology
Laboratory, School of Biotechnology, JNU. I thank him for his invaluable time and constant
motivation despite of his busy schedule. I humbly like to acknowledge Dr. Rita Sharma. Her
profound scientific approach helped me to a greater extent.
I thank my professors Dr. Sonika Gupta, Dr. Anandita Singh, Dr. Sitaraman Ramakrishnan,
Dr. Pallavi Somvanshi, Dr. Prateek Sharma, Dr. Chaitanya Madhurantakam, Dr. Sonika Gupta
and Dr. Udit Soni, Department of Biotechnology, TERI University for enlightening me to the
path of research.
I would like to thank the laboratory members, Vinay Singh, Vasudha Bharadwaj, Supriya
Mathur, and Neeraj Kumar who has helped me to learn a lot of new things, due to the
frequent discussions which provided the deep insight of the subject. I am thankful to my
fellow mean Sushree Sangita Priyadarshini for being supportive while carrying out the
experiments.
I will remain indebted to Mr. Satybeer for his technical assistance.
Lastly, I would like to express my sincerest thanks to my parents for their blessings and
constant encouragement.

Abbreviations

Degree Celsius

Microlitre

Microgram

Ng

Nanogram

OD

Optical Density

PCR

Polymerase Chain Reaction

QPCR

Quantitative PCR

rpm

Revolutions per minute

min

Minutes

Hr

Hour

MQ

Milli-Q

sec

Second

cDNA

Complementary DNA

RNA

Ribonucleic Acid

TAE

Tris Acetate EDTA Buffer

DEPC

Diethylpyrocarbonate

DNA

Deoxyribonucleic acid

EDTA

Ethylenediaminetetraacetic Acid

NaCl

Sodium Chloride

Ct

Cycle threshold

Tm

Melting temperature

Sb

Sorghum bicolor

CONTENTS
CONTENTS

Chapter 1: Introduction
Chapter 2: Review of Literature
2.1 Normalization
2.2 Reference Genes
2.3 Sorghum
2.4 Scope of the study
Chapter 3: Materials and Method
3.1
Materials
3.1.1
Reagents
3.1.2
Plant Material
3.1.3
Miscellaneous
3.2
Methods
3.2.1
MS Media Preparation
3.2.2
Seed Germination
3.2.3
RNA Extraction using Trizol reagent
3.2.4
Agarose gel electrophoresis
3.2.5
cDNA synthesis
3.2.6
Selection of candidate genes
3.2.7
Primer designing and expression analysis using Q-PCR
3.2.8
QPCR Reaction
Chapter 4: Results
4.1
Plant Material
4.1.1
Plants in the field
4.2
RNA Extraction
4.3
Agarose gel electrophoresis
4.4
cDNA Synthesis
4.5
Primer Designing
4.6
Q-PCR based analysis
4.7
Normalization
Chapter 5: Discussion
References
Annexure

8
10
13
14
16
16
17
17
17
17
17
18
18
18
18
19
19
19
20
20
21
21
21
21
22
23
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25
26
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29
30

ABSTRACT
Sorghum bicolor is a C4 grass that belongs to Poaceae a grass family and is one of the most
economically important cereal crops. It is grown worldwide for food, feed and biomass. In the recent
years, Sorghum has emerged as a model system to study, understand and improve C4 crops, and also
to gain insights in carbohydrate partitioning. Gene expression studies play an important role in
characterising gene function and Quantitative Real-time PCR (qPCR) serves to be one of the robust
methods for this purpose. Reference genes are used to normalize the mRNA levels among different
samples to rule out the variations caused by technical sample handling. Most often constitutively
expressing genes Housekeeping Genes are used as for this purpose. It has been found that
expression of housekeeping gene may vary among different plant species and therefore identification
of species-specific genes whose expression is stable, is critical for qPCR based expression analysis.
Objective of this work is to identify putative reference genes for sorghum based upon the literature
survey and evaluate the selected candidates to characterize their expression stability in different plant
organs and among various cultivars of sorghum. Based upon the literature survey we have selected
five genes (Glyceraldehyde 3-phosphate dehydrogenase, -actin, eukaryotic initiation factor-5, tubulin, Ubiquitin). Gene-specific qPCR primers were synthesized and used to check their expression
in seedlings of several sorghum cultivars and various organs like leaf, root and stem of sorghum
cultivar M35-1. We have used delta-deltaCt method to compare the relative gene expressions. The
selected genes have been ranked according to their expression stability (high to low) in different
samples.

Keywords: Q-PCR, Reference Genes, Normalization, Endogenous control

INTRODUCTION
Morphological traits are inherited by the ancestors, which are conveyed by some factors
called genes. Genes are the hereditary material, which one inherits from its ancestor. That is
why it is also known as the unit of inheritance. A gene gives rise to a phenotype by
generating an RNA molecule or a protein product. Thus, the functional genetic unit should
not only surround DNA that is transcribed into RNA but also all the components which are
involved in its transcription.

Figure. 1.1 Genotype giving rise to the phenotype

The expression of a gene varies in response to various environmental conditions and can be
used to understand role of a gene product in the metabolic pathways and their interrelations.
It is one of the most important fundamental levels at which the genotype gives rise to a
phenotype.
Several techniques have been developed to analyse gene expression and its quantification.
These include Northern blotting, quantitative real time PCR (qPCR), Serial Analysis of Gene

Expression (SAGE), microarrays and transcriptome sequencing. Northern Blotting is one of

the traditional techniques and is used to detect the particular RNA transcript. High throughput
techniques like Microarrays, RNA sequencing etc.are used to generate whole genome
transcriptome patterns. However, for the expression quantification of one or a few genes,
qPCR is an alternative method of choice. It helps to monitor the amplification of DNA in real
time. It is one of the robust techniques that can be used to determine the low levels of mRNA
transcripts with great sensitivity.
When using qPCR for expression quantification among several samples or various replicates
of same sample, it is very critical to consider the variation caused during the technical
handling of the samples like amount of tissue used, RNA quality, RNA quantity, cDNA
synthesis efficiency etc. In order to nullify such variations in the samples, endogenous
controls for normalization of expression levels are used. These endogenous control genes are
called as reference genes. Major feature of the reference genes is that their expression should
not vary between different samples or in response to experimental conditions.
Usually, housekeeping genes falls under this category. It has been found that expression
levels of even housekeeping genes is not stable across species e.g. GAPDH shows an
unregulated expression in roots of Saccharum officinarum but not in the leaves. Therefore
identification and validation of species-specific reference genes is critical before using them
for gene expression quantification. Objective of this study is to quantify the expression of
putative reference genes during various plant organs of Sorghum and identification of
reference genes that shows least variation among sample types.

Objectives:
1 Reference gene selection and designing primers
2 RNA Isolation and cDNA synthesis.
3 Expression analysis of the selected reference genes using Q-PCR.
4 Data Validation using different statistical algorithms.

Chapter 2
REVIEW OF LITERATURE
Genes are defined as the basic structural and functional unit of inheritance. Genes are
composed of DNA and they encode proteins that control the functions of a cell. A eukaryotic
genome has thousands of genes that are expressed in a cell-specific manner. Fate of a cell or
an organ of an organism is determined by the type of genes that are expressed. Gene
Expression can be defined as the process where the information stored in the genes in the
form of DNA is translated to synthesize functional gene products. Several small-scale and
large-scale approaches are available to study the gene expression. Though the modern
technologies to study large-scale gene expression have revolutionized the world of genetics,
small-scale expression studies have long and rich history, and are very important for
functional genomics studies. PCR-based detection technologies have proved to provide an
indispensable information regarding the plant/environment interaction (Deepak et al. 2007).
PCR was discovered by Kary Mullis and colleagues in 1983 (Saiki et al, 1988) and is one of
the earliest techniques that empowered scientists with robust methods to study cellular and
molecular processes (Walker- Molecular Biology and Biotechnology 4e RSC, 2000).

Figure 2.1 Stages and Components of PCR

PCR consist of three steps, namely:

a) Denaturation where DNA is heated up to separate the two strands
b) Annealing where primers bind to single stranded DNA, and,
c) Extension where polymerase synthesizes the complementary DNA strands to form
amplified form of the double stranded DNA under study.

Figure 2.2 Steps of PCR

https://en.wikibooks.org/wiki/Structural_Biochemistry/Polyermase_Chain_Reaction/How_PCR_is_
Performed

It doubles the amount of target DNA and this cycle is repeated many times, thereby leading to
amplification of target DNA sequence many fold. In the process, millions of copies of that
fragment are synthesized.
Real-time PCR or Quantitative-PCR is one of the most sensitive, powerful and reliable tool to
quantify gene expression. In the past decades it has been used for a variety of applications
like gene expression, copy number variations, quantification of pathogen load, etc. (Raso,
.A. Roberto Biassoni, .R. 2014).Traditional PCR works on the principal of end-pint detection
where amplified DNA is usually analysed by agarose gel electrophoresis. Whereas in qPCR,
fluorescent dyes are used in addition to the general PCR reaction components, which help in
the quantification of total DNA as PCR amplification occur. This makes it possible to
measure the initial concentration of DNA in the reaction. Over the years several fluorescent
chemistries have been discovered but the most common fluorescent reporter molecules used
in qPCR are SYBR Green Dye or TaqMan Probes(Life technologies 2012).SYBR Green is a
fluorescent dye that can bind preferentially to any double stranded DNA molecule. It

intercalates in the minor groove of an amplified product. DNA-SYBR-Green Complex

absorbs the blue light at 497nm and emits the green light at the 520 nm. SYBR green binds to
the DNA and excited SYBR bound to DNA gives a strong florescent signal as compared to
the unbound SYBR (Figure 2).

Figure 2.3 Mechanism of Flourescence detection using SYBR green dye

(http://www.biorad.com/featured/en/sybr-green-for-qpcr.html)

TaqMan probes are the hydrolysis probes of variable lengths (oligonucleotide), which bind to
the single stranded DNA based upon the complementarity between the probes and the target
DNA sequence. They are specially designed to enhance the specificity of qPCR studies.
TaqMan probes are based on the principle of 5-3 exonuclease activity of Taq polymerase,
which cleaves the dual-labelled probe when gets hybridized to the complementary DNA.
TaqMan probes consist of:
a) A fluorophore, attached to the 5` end of the probe
b) A quencher, attached to the 3` end of the probe
The quencher reduces the emissions of fluorophore molecule, when present in the close
proximity with a fluorophore via FRET (Forster Resonance Energy Transfer).
During the reaction, TaqMan probes and the primers bind to the DNA. TaqMan probes bind
between the binding sites of the primer pair (Fig. 3A) Taq polymerase extends the primers to
synthesize the daughter strand (Fig. 3B). The 5-3 exonuclease activity of polymerase
cleaves the probe (if hybridized to a correct target) (Fig. 3C). Once, the probe gets cleaved
off, it moves away from the quencher molecule. Released fluorophore is no longer in the

proximity of the quencher molecule and hence, fluorescence signal is produced by the
fluorophore.

Figure 2.4 Pictorial representation of one PCR cycle using TaqMan Probes
A. Primers and TaqMan probes bind to the template DNA, B. Polymerase start synthesizing the
complementary strands and upon contact with TaqMan probe oligo, displaces, it. C. Fluorophore
is released from the quenching effects of the Quenchers molecule and D. Completion of the PCR
cycle doubles the template DNA. (http://www.dkfz.de/gpcf/lightcycler480.html)

2.1 Normalization
Normalization is a major step in the quantification of a gene expression analysis
(http://normalisation.gene-quantification.info/). Endogenous controls can enhance the
reliability of the data generated during qPCR, by eliminating the errors which may occur due
to technical variations. Hence, there is a need to an accurate and reliable normalisation. These
endogenous control gene are called as reference genes.

Many technical variations can lead to erroneous results. These variations may include:

Amount of starting material;

RNA quality;
cDNA synthesis efficiency;
In proportionate addition of the reagents due to pipetting, etc.

Therefore, in order to neutralize the variation of gene expression due above mentioned
factors, accurate and reliable normalization is critical. For this purpose, endogenous genes
whose expression is stable across tissue organs, various developmental stages or in response
to various environmental conditions are required.
2.2 Reference Genes
Reference genes are the endogenous controls which are used to normalise the variations that
can occur due to technical errors. To use any gene as a reference gene it must meet the
following criteria
i
ii
iii

Stable expression across the tissues, organs and various developmental stages.
Their expression should not be affected by the environmental conditions,
to avoid gDNA amplification, it should not be associated with any pseudo genes,

Ct values should not be very low (<15) or very high ( >30) (Wan et al. 2010).Housekeeping
genes that are involved in the basic metabolism of the cell are good candidates as reference
genes. During last two decades several genes have been characterized as reference genes.
Though expression of these genes is considered to be constitutive, but practically it is not
true. Housekeeping genes may show variability in their expression levels in different tissues.
(Selvey et al. 2001; Vandesompele et al. 2002; Ohl et al. 2005; Rebouas et al. 2013).
Variation in their expression levels among different species have been reported in the
literature. Several species like Saccharum officinarum, Zea mays, Oryza sativa etc. were
studied for the characterization of the reference genes and was found that the reference genes
are species specific. What seems to be perfect in one species, does not guarantee its
functionality in another one. For example, GAPDH (Glycerol-3-phosphate dehydrogenase)
has proven to be the stably expressed gene in the genome of Saccharum officinarum(Silva et
al. 2014). But, in Zea mays, -tubulin and eIF-1 showed the stable expression across the
genome (Zhang et al. 2014).

Table 2.1: List of some of the reference genes, which are commonly used to normalize
the gene expression in different species.
Sl.
No.

Species

ReferenceGene

Author

Saccharum

GAPDH,TUB,H1

Panicum
virgatum

UBQ5,CYP5,U2AF,FTHS4,eIF4,eEF1

Fescue

GAPDH,Tub,TIP41,CACS,SAND
(ATG28390),ACT,Fbox(AT5G15710),
PEPKR1

Yangetal.2015

Fagopyrum

CACS(AT5G46630),TIP41,ADH3,G6PD

Nataliaetal.2011

Arachis

CYP2,ACT11,ELF1B

Reddyetal.2013

Cajanus

GAPDH,UBC

Sinhaetal.2015

Saccharum

GAPDH,eIF4,CACS,CUL

Lingetal.2014

Hordeum

GAPDH, TUB, miR159, ACT, ADP

Ferdousetal.2015

Lolium

GAPDH,eEF1A,eIF5A,TBP1,YT521

Wangetal.2015

10

Zeamays

GAPDH,TUB,CYP,EIF4A,EF1A,ACT2

Silvaetal.2014
Gimenoetal.2014

Linetal.2014

Various statistical algorithms are being used to analyse the gene expression stability namely
GeNorm, NormFinder, RefFinder, BestKeeper etc. These approaches uses the excel
workbook to assess the multiple genes. GeNorm calculates the pairwise variation of a
reference gene with the other candidates (Rebouas et. al, 2013). The gene stability measure
M is defined as the pairwise variation of a reference gene w.r.t the other candidate genes.
Lower the M value, more stable is the expression of a gene. Its freely available
(http://medgen.ugent.be/ ~jvdesomp/genorm/) Similarly, BestKeeper calculates the Ct values
of all the genes with stable expression levels and are being grouped. The one with less
standard deviation (<1) are considered to be the stable ones. The use of these normalization
tools, can provide us the stability analysis of the reference genes in different type of samples
(Rebouas et. al, 2013)It is good to use the combination of reference genes for normalization,
since one gene can give rise to the large errors. Hence, the use of two reference genes or a
combination of a few genes would give an accurate result (Vandesomple et. al, 2002; Gimeno
et al. 2014)
2.3 Sorghum

Sorghum is a hardy and robust crop, which grows particularly in Semi-Arid Tropical regions.
It is a C4 crop with high photosynthetic efficiency. Based upont the usage, sorghum has been
classified into Grain sorghum, Energy Sorghum, forage Sorghum and Sweet Sorghum. Grain
sorghum is one of the most staple foods for the millions of the poor people and serves many
health benefits as it is anti-diabetic in nature, can reduce the levels of cholesterol etc. Sweet
sorghum accumulate fermentable sugars in its stems and is a potential bioenergy crop.In
1970s, Nimbkar Agricultural Research Institute situated in Maharashtra started working on
Sweet Sorghum. It is said to be a dual-purpose crop as it is a hybrid of two lines with a high
yield of grain and stem biomass respectively. Sweet Sorghum is being studied for 3 main
reasons i.e.
a) Bioenergy crop
b)C-partitioning
c) Health benefits
Sweet Sorghum can be used as a livestock under the hot and dry climatic conditions
(Almodares et al., 2009). The by-product of this crop (i.e. bagasse) can be used to generate
the heat energy, manufacturing of paper, silage for the animal feed, fibre for the ethanol
production etc.

2.4 Scope of the study

The objective of this study is to identify the expression patterns of various housekeeping gene
in various tissue organs of sorghum. In addition, expression of these genes will be analysed in
the seedlings of various sorghum cultivars. Till date, expression profiling to identify the
sorghum specific reference genes has not been identified. This study will result in
identification of most stable reference genes in sorghum than would help in gene expression
studies in Sorghum.

Chapter 3
RESOURCES AND METHODS
3.1 MATERIALS
3.1.1 Reagents
Trizolreagent (Ambion, Life Technologies), Chloroform, Isopropanol, Ethanol (Emsure ),
cDNA synthesis kit (Agilent Technologies), SYBR Green/Evagreen Mastermix (Agilent
Technologies), Nuclease free water (Amresco), Agarose (Pronastar, Agarose Plus), Tris
Acetate EDTA Buffer (TAE) prepared in Diethylpyrocarbonate (DEPC) water, Red Safe Dye
(Beijing SBS Genetech co. pvt. Ltd.), 1Kb DNA Ladder (Br. Biochem), DNA Loading dye,
Electrophoretic unit (Bio-Rad)
3.1.2. Plant Material
Several varieties/germplasms for Sorghum bicolour were used in this study and are listed in
Table 3.1. Seeds for these germplasms were obtained from Dr. Umakant, Indian Institute of
Millet Research, Hyderabad.
Table 3.1List of sorghum varieties used in this study for expression analysis
S. No.
1
2
3
5
6
7

Cultivar Name

M35-1
CSH30
CSV15
CSV22
CSV32 F
SSV84

Hybrid/variety
Variety
Hybrid
Variety
Variety
Variety
Variety

Type

Grain Sorghum
Kharif Sorghum
Kharif Sorghum
Rabi Sorghum
Forage Sorghum
Sweet Sorghum

3.1.3. Miscellaneous
Pestle-mortar, MCTs, Micro tips, PCR tubes, MQ Water, Ethanol.

3.2 METHODS
(The detailed protocol of all the media and solutions used is mentioned in the Annexure)
3.2.1 MS Media Preparation
Murashige-Skoog (MS) media was used for growing sorghum seeds. Detailed composition of
the MS media is listed in Table: A1.
3.2.2 Seed Germination
Seeds were soaked in tap water for 4-6 hours and water was replaced with fresh water every
30 minutes before sterilization in the laminar air flow. Soaked seeds were washed with Milli
Q water and then rinsed with 70% ethanol for 30-40 seconds with constant shaking. Again
seeds were again rinsed with MQ water. Seeds were then treated with 4% sodium
hypochlorite (NaOCl) for 12-13 minutes with intermittent shaking. Seeds were then washed
with autoclaved MQ water 4-6 times, for 2-3 minutes each. After blotting the residual water
on the autoclaved Whatmann paper, seeds were put on the MS basal medium in the jam
bottles for germination and incubated these jam bottles at 28Cwith light intensity of
110.295mol m-2sin the plant tissue culture room. For harvesting mature leaves, internode,
nodes and root samples, M35-1 seeds were germinated on soil.
7-10 days old seedlings of M35-1, SSV84, CSV32F, CSV15, CSH30were harvested and
stored at -80C till further use. From about two month old M35-1 plants grown in the field,
mature leaves, nodes, internodes and root samples were collected and stored at -80C till
further use.
3.2.3 RNA Extraction using Trizol reagent
About 100 mg tissue (leaf/roots/stem)was ground to fine powder using mortar-pestle chilled
in liquid nitrogen followed by the addition of the Trizol reagent (1 ml per 100 mg of tissue).
The contents were transferred to the 2 ml micro-centrifuge tube and were allowed to sit for 10
minutes. 200lof chloroform was added and mixed gently. Contents were allowed to settle for
10 minutes followed by centrifugation at 13,000 rpm at 4 0C for 10 minutes. Amongst the 3
layers obtained, top aqueous layer was collected and was transferred to the clean microcentrifuge tube, followed by the addition of 0.6 ml of isopropanol (per ml of Trizol reagent).
Again, the contents were centrifuged at 13000 rpm at 4 0C for 10 minutes. The supernatant
was decanted and the pellet was washed with 70% ethanol. RNA pellet was dried under the
laminar hood and dissolved in 40l of RNase free water. The concentration and quality of the
dissolved RNA was measured using the Nano-spectrophotometer (Nanodrop 2000, Thermo

scientific), using Nuclease free water as reference. One l of RNA sample was used to check
its absorbance. Quality was determined by the OD260/OD280 ratio.
3.2.4 Agarose gel electrophoresis

Figure: 3.1 Electrophoretic Unit

(https://www.addgene.org/plasmid-protocols/gel-electrophoresis/)

Electrophoretic Unit (Bio-Rad)was used to perform agarose gel electrophoresis. Various

components of electrophoresis unit are shown in Figure 1. Agarose gel (0.8%) was prepared
(Table A3) and 4l of Red safe dye was added to the gel to allow visualisation of RNA. Gel
was poured in the casting tray with the comb fixed in it. After the gel solidified, combs were
removed and the casting tray containing the gel was put in the buffer tank containing ~600ml
of 1X TAE buffer. Four l of 1Kb DNA ladder (Thermo Scientific)wasused as size marker.
Electrophoresis was performed at 120 mV. After electrophoresis, gel imaging was performed
using the Gel Doc (Bio-Rad).
3.2.5 cDNA synthesis
Affinity Script, First strand cDNA synthesis kit (Agilent technologies) was used for cDNA
synthesis. Three microgram of RNA was used for cDNA synthesis in a 100 l PCR tube. Kit
components were thawed on ice and added to the PCR tubes as described in table A4
3.2.6 Selection of candidate genes
Available literature was surveyed to identify gene characterized as reference gene from other
plants species. Five genes showing stable expression in monocots were selected for
expression analysis in sorghum.

3.2.7 Primer designing and expression analysis using Q-PCR

Coding DNA sequences of candidate genes were retrieved from Phytozome database. The
sequences were searched for 150-300bp unique regions. The unique regions were determined
using Basic Local Alignment Search Tool (BLAST) against the Sorghum genome. The
regions with the best hit were selected. Primers were designed for 75-150bp long
amplicons.Primer3online toolwas used for the designing the primers. Specificity of the
primers was checked by using BLAST search on sorghum genome on PHYTOZOME
database. The e-value threshold was increased to 5 while performing the BLAST search
3.2.7 QPCR Reaction
SYBR Green dye (Agilent technologies) was used for Q-PCR. The components of Q-PCR
were thawed on ice and were mixed in the PCR tubes (Table A.5). Reaction with all the
components without cDNA template was used as negative control. Mastermix were prepared
for various components of the QPCR reaction and distributed evenly to each qPCR reaction
optical strips. Nuclease free water to make up the volume to 20l.One l of experimental
cDNA was added to each single reaction mixture followed by gentle mixing. A short flash
centrifugation was given to the reaction mixture. Then, the optical qPCR tube strips were put
in the instrument. Appropriate PCR program (Figure 3) was selected and the thermal profile
was set up according to the requirement. For cDNA synthesis, recommended concentrations
of genomic DNA and cDNA are 5 pg 50 ng and 0.5 pg 100 ng, respectively.

Figure 3: Thermal setup for qPCR reaction

Segment 1 represents the initial denaturation. Segment 2 represents the 40 cycles of
amplification where each cycle consist of denaturation, annealing and polymerisation.
Segment 3 represents the melt curve analysis setup.

Chapter 4
RESULTS
4.1 Plant Material
Sorghum seeds were germinated on the MS basal medium, under aseptic conditions (Figure
). Seven days old seedling (Figure . . . .) were harvested and froze in liquid nitrogen.

Figure . . A and ..B

For the collection of tissue samples for various plant organs like leaf, stem internodes, stem
nodes, panicle, seeds, roots etc, sorghum seeds were planted on the field. Figure 4.2 (A) and
(B) shows seven weeks old M35-1 plants grown in the field conditions. Leaf tissue, stem
tissue and roots have been harvested from two month old M35 sorghum plants.

4.1.2 Plants in the field

(A)

(B)

Figure 4.2 Field grown sorghum plants.

Sorghum plants for various cultivars were grown in this field condition. (A) About seven weeks old
M35-1. (B) About seven week old CSV24SS plants.

4.2 RNA Extraction

Seedlings of M35-1, SSV84, CSV32F, CSV15, CSH30 and mature leaves, nodes, internodes
and roots were used for RNA extraction using the Trizol reagent. About 100mg tissue was
used for isolating the RNA. The concentration and absorbance ratio (OD 260/OD280) measured
by the Nano-spectrophotometer is mentioned in the table 4.1
Table 4.1 Nanodrop Spectrophotometer reading of isolated RNA
S.
No.

Variety

Sample

Concentratio
n (ng/l)

A260

A280

A260/280

A260/23
0

CSV32F

Seedling

1370.5

34.263

16.76

2.04

1.40

2.04

1.89

8
2

CSV15

Seedling

3674.8

91.870

45.14
2

CSH30

Seedlings

946.4

23.660

11.438

2.07

1.25

SSV84

Seedlings

1435.4

35.886

17.62

2.04

1.36

2.01

1.71

8
5

M 35-1

Leaf

4028.2

100.70

50.12

M 35-1

Node

1880

46.99

22.99

2.04

1.44

M 35-1

Internode

329.1

8.2

3.8

2.15

0.92

M 35-1

Roots1

2396.5

59.913

29.64

2.02

1.69

2.05

1.48

0
9

M 35-1

Roots2

1095.8

27.394

13.34
2

4.3 Agarose gel electrophoresis

To check the quality and integrity of the isolated RNA samples, we used 0.8% agarose gel for
electrophoretic separation. After the gel electrophoresis, gel imaging was done under the Gel
Doc. Two distinct bands of 25S rRNA and 16S rRNA was observed. As shown in Figure 4.3.

A.

B.
1Kb

1Kb

B1 B2 B3 B4 B5

A1 A2 A3 A4

Figure 4.3 Agarose gel electrophoresis of RNA samples

0.8% agarose gel was used for electrophoretic separation of RNA samples. A). RNA extracted
from seedling of various sorghum cultivars. Lane A1, A2, A3, A4 represents CSV32F, CSV22,
CSV15, CSH30 respectively. B). RNA extracted from various plant organs of M35-1
plants.Lane B1, B2, B3, B4, B5 represents the RNA sample from leaf, node, internode, root 1,
root2respectively.

4.4 cDNA Synthesis

Three microgram of total RNA was used for the preparation of cDNA. cDNA synthesis
was performed by using the Affinity Script, First strand cDNA synthesis kit (Agilent
technologies). For each sample 20l cDNA synthesis reaction ion was performed. After
cDNA synthesis, each sample was diluted three times and 1l of this diluted sample was used
for each reaction.
Gene Selection:

Based upon the literature survey, we prioritize ten genes were identified that can be used as
reference gene for normalization in QPCR based gene expression analysis. These gene
are . . . .. Top five genes were selected for expression analysis.

4.5 Primer designing

cDNA sequence of selected candidate genes were retrieved from Phytozome and used to
design primers for SYBR Green expression analysis. Primers were designed using the primer
designing tool Primer 3. Primer sequences with their features like length, Tm, GC content etc.
are listed in table 4.2.
Table 4.2 Primer Information
Annotati Primer Name
on Name

Primer Sequences

Prim Tm
er
Leng
th

GC
(%)

Prod.
Size

Prod. Tm

qSb_actin_F
Actin-1

ACAATCGGTGCAGAAAGGTT

20

59.6

45

85

77.9

CTTCATGGATGCCAGGAGAT

20

60.03

50

85

77.9

ACAGGCATTCTTTGGCTCAC

20

60.26

50

85

72.8

TCTTTCAGCAAGGCTTTCGT

20

60.13

45

85

72.8

AACGACCTCGTCTCCGAGTA

20

59.87

55

91

83.9

CCTGGAGGTCTCCTTCCTCT

20

59.8

60

91

83.9

GTTGCAGCTGGAAAGGACTC

20

60

55

88

80.6

CGTCCTCTTCCTCAGACTCG

20

60.13

60

88

80.6

qSb_actin_R
Actin-1
qSb_ub_F
Ubiquitin
qSb_ub_R
Ubiquitin
qSb_tub_F
Tubulin
qSb_tub_R
Tubulin
qSb_eIF_F
eIF-5
qSb_eIF_R
eIF-5

qSb_gapdh_F
GAPDH

TCCACAGACTTCGTTGGTGA

20

60.29

50

93

79

CCACGAGACAAGCTTGATGA

20

59.98

50

93

79

qSb_gapdh_R
GAPDH

4.6 Q-PCR based Expression Analysis

Primers for the selected five genes were designed as discussed. To check the specificity of the
primers, QPCR reaction with cDNA was done, to check the specificity of primers.
Table 4.3Alignment of the samples along with their Ct Values
1

10

A Mleaves MInternode MRoot2 CSV32F CSH30 Mleaves MInternode MRoot2 CSV32F CSH30
Ct: 23.76

Ct: 23.36

Ct: 31.90

Ct: 28.77

Ct:23.12

Ct: 29.56

Ct: 28.03

Ct:

Ct:

Ct: 27.97

B Mleaves MInternode MRoot2 CSV32F CSH30 Mleaves MInternode MRoot2 CSV32F CSH30
Ct: 23.86

Ct: 22.99

Ct:31.81

Ct: 27.85

Ct: 23.88

Ct: 30.47

Ct: 29.95

Ct:

Ct:

Ct: 26.06

C Mleaves MInternode MRoot2 CSV32F CSH30 Mleaves MInternode MRoot2 CSV32F CSH30
Ct: 23.49

Ct: 23.09

Ct: 31.12

Ct: 26.22

Ct: 23.22

Ct: 29.76

Ct: 31.56

Ct:

Ct:

Ct: 25.64

D Mleaves MInternode MRoot2 CSV32F CSH30 Mleaves MInternode MRoot2 CSV32F CSH30
Ct:: 23.75

Ct: 22.74

E MNode

MRoot1

Ct: 23.19

Ct: No Ct

Ct: 31.91

Ct: 29.85

Ct:28.56

Ct:

SSV84 CSV15 NTCeIF MNode

MRoot1

SSV8

Ct: 33.10

Ct: 22.75

Ct: 27.24

Ct: 23.50

Ct: 23.43

Ct: 32.08

Ct: 26.52

Ct:

Ct: 26.19

CSV15 NTCTub
Ct: 26.43

Ct: 36.13

Ct: 25.88

F MNode

MRoot1

Ct: 22.39

Ct: 28.91

SSV84 CSV15
Ct: 23.43

Ct: 23.36

MNode

MRoot1

SSV8

CSV15

Ct: 26.05

Ct: 31.29

Ct: 26.85

Ct: 25.91

G MNode

MRoot1

Ct:: 22.82

Ct: 29.58

SSV84 CSV15
Ct: 23.80

Ct: 23.13

MNode

MRoot1

SSV8

CSV15

Ct: 26.16

Ct: 32.14

Ct: 26.04

Ct: 25.07

H MNode

MRoot1

Ct: 23.29

Ct: 28.22

SSV84 CSV15
Ct: 23.07

Ct: 23.51

MNode

MRoot1

SSV8

CSV15

Ct: 25.83

Ct: 31.36

Ct: 26.30

Ct: 26.26

A.

B.

Figure 4.4 Pictorial representation of QPCR amplification plots and Melting curve plots
Amplification plots and Melt curve plots were obtained from QPCR Machine. Figure (A) and(C) represents the
amplification plots of genes eIF-5 and -tubulin respectively. Figure (B) and (D) represents the dissociation
curves obtained from melting curve analysis of amplicons of eIF-5 and -tubulin respectively.

4.6.2 Normailization
For the relative quantification of the expression of tubulin gene, we have used eIF-5 as a
reference gene and vice versa. However when data for these genes from various biological
replicates and all the organs, developmental stages or stress conditions will be available, we
will use the statistical algorithms like GeNorm, NormFinder, BestKeeper, RefFinder etc to
identify the most stable gene across sorghum organs and various cultivars.
Ct was calculated by using the Ct values of all the samples.
1. Normalize Ct-Test with reference to CT-Ref
Ct (Test) = Ct (Test) CT (Ref)
2. Normalize Ct-Test with CT-Caliberator
Ct = CT-Test Avg CT-Caliberator
3. Fold-Change
Fold change= 2- Ct
Therefore, in this case Reference is the Mleaves and the calibrator is the MRoot2

(A)
Differential expression of -tubulin
1.6
1.4
1.2
1
0.8
0.6
0.4
0.2
0
MN

MI

SSV84

CSV15

CSH30

(B)
Differential expression of eIF-5
1.8
1.6
1.4
1.2
1
0.8
0.6
0.4
0.2
0
MN

MR1

SSV84

CSV15

CSH30

Figure 4.4 Relative expression levels of tubulin and EIF genes.

Fold change of the expression level of the reference genes A. Elongation factor and B. Tubulin,
in different samples. MN, Mature node; MR1, Mature root, SSV84, seedlings of variety
SSV84; CSV15, seedlings of variety CSV15; CSH30, seedlings of sorghum hybrid CSH30.

Chapter 5
DISCUSSION
Sorghum is the versatile anda hardy crop, belongs to the grass family. It is a first C4 plant
whose whole genome sequence is available, which allows us to study the C4 pathway
evolution from a C3 progenitor (http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2718502/).
Sorghum genus has huge genetic diversity. Whereas grain sorghum or energy sorghum has
large biomass, sweet sorghum cultivars store fermentable sugars in their stems. This diversity
of presence of structural carbohydrates and soluble sugars make it a good model system to
study the carbon partitioning.
For QPCR based gene expression studies, identification of reference genes is critical. Several
species have been studied for reference gene identification like Hordeum vulgare, Oryza
sativa, etc. Also It has been found that any one gene may not be used as reference gene across
species or genus as its expression may change in a species-specific manner. For example, 18S
rRNA was found to be the most reliable gene for normalization in Rice (Jain et al. 2006; Kim
et al. 2003). Whereas, in Hordeum vulgare ADP was found to be the most stable one. The
reference genes are used as a normalizing factor in the Q-PCR based expression analysis, as
their expression is considered to be the stable one. However, this is not a universal
phenomenon and expression of these genes may vary in response to various environmental
conditions (Jain et al. 2006). The reason for their varied expression can partly be explained by
the fact that they not only helps in the basic metabolism of a cell, but also in the cellular
function (Jain et al. 2006). Over the last two decades, the studies related to the identification
of reference genes in many plant species has been performed (Gimnez et al. 2010; Hong et
al. 2008; Tong et al. 2009; Lee et al. 2010) and it is advisable to identify species-specific
reference genes for gene expression studies.
Q-PCR is a robust technique to analyse the expression of a gene. To get the accurate results,
normalization to rule out the variation caused by technical handling is critical. We surveyed
the literature and identified several candidate genes that can be used as reference gene in crop
plants. we prioritized these genes according to their stability across species and selected top
five reference genes. These five genes are GAPDH, -tubulin, eIF-5, Ubiquitin and Actin1.We do not have any universally accepted method for the identification of reference gene

with a stable expression (Ferdous et al. 2015), NormFinder, BestKeeper or GeNorm, which
are the statistical algorithms are used. Because of plants growth cycle and time constraints we
could analyse the expression of only two genes in a few sample (i.e. eIF-5 and tubulin).
Ct method was used to analyse the data. Further sampler harvesting, QPCR and data
analysis is in progress.

ANNEXURE
Table: A.1. Components of MS Media Basal
S. No.

Components

Volume

MS Media

4.46gm

Sucrose

30.0 gm

Agar

4.0 gm

Distilled Water

1000 ml

Note: pH was set at 5.8 (before adding the agar)

Table: A.2. Components of 1X TAE

S. No.

Components

Concentration

Tris

40mM

Acetic acid

20mM

EDTA

1mM

Table: A.3. Components of Agarose Gel

S. No.

Components

Volume

Agarose

0.8gm

1X TAE

100 ml

Red Safe/Good View Dye

4 l

Table: A.4. Components of cDNA synthesis

S. No.

Component

Volume (l)

Mastermix

10

Random Primers

Enzyme

1.6

RNA

Nuclease free water

Table A.5Components for the Q-PCR reaction mixture

S. No.

Components

Concentratio
n (nM)

Volume (l)

2x SYBR green
QPCR Mastermix

10

Forward primer

200

Reverse primer

200

Reference dye

30

0.3

Nuclease free
water