RNA Lab
(Isolation, quantification and
qPCR analysis)
MCB7300
Work Flow
Day 1. RNA isolation from Arabidopsis leaves
Day 1. RNA quantification in Nanodrop and
Bioanalyzer analysis
Day 1. Reverse transcription to cDNA
Day 2. Setting up for qPCR
Day 2. Data analysis
Method of evaluation
• You will have an in class quiz worth 50
points on Wednesday (Feb. 18th) and a
lab report worth 50 points which must
be turned in by next Wednesday (i.e.,
Feb. 25).
• Please submit a hard copy of your lab
report.
RNA Isolation of leaves of Arabidopsis
1. What is the maximum amount of starting material?
100 mg
2. Is the yield of total RNA the same for the same amount of starting material for
different plant species?
No, the yield varies for different plant species.
3. Which lysis buffer can be used for plant materials?
 Buffer RLT (Guanidine Isothiocyanate) is used for all tissues except
endosperm and tissues containing endosperms (e.g., seeds).
1. Buffer RLC (Guanidine Hydrochloride) is used for seeds with endosperm
4. Is total RNA isolated with RNeasy kit free of genomic DNA?
No, most (but not all) of DNA is eliminated. Therefore, if total RNA will be used
for downstream application such as Reverse-transcription-PCR (RT-PCR), then DNasetreatment must be carried out for the total RNA.
5. What is the role of QIAshredder homogenizer?
It simultaneously removes insoluble material and reduces the viscosity of the
lysates by disrupting gelatinous material.
Quality check for RNA samples
• UV/VIS Ratios
– Absorption 260/230 ratio ≥ 1.0 and 260/280 ratio ≥ 1.8
– Low 260/280 ratios are often attributed to phenol and/or protein
contaminations.
– Low 260/230 ratios are usually attributed to salt (e.g. guanidine isothiocyanate)
and/or phenol contaminations.
– “High-salt”, seen as 260/230 ratio less than 1.0,
• Bio-analyzer
–
RIN (RNA integrity) ranges from 1 to 10, with 1 being the most degraded profile and 10
being the most intact.
• Gel Electrophoresis
–
RNA sample integrity can also be evaluated using one of several standard denaturing gel
electrophoresis methods.
Quantification of RNA samples by Nanodrop
Quality check for RNA samples
RIN 9.2
RIN 6.2
RIN 3.2
http://itghumangenomeprojectwallpapars.blogspot.com/2012
/12/agilent-bioanalyzer.html
Real-Time qPCR
• Real‐time qPCR is the most sensitive and reliable
method for detection and quantification of nucleic
acids (DNA, cDNA, & RNA) levels.
• It is based on detection and quantification of
fluorescence.
• Emitted from a reporter molecule at real time.
• This detection occurs during the accumulation of the
PCR product with each cycle of amplification, thus
allows monitoring the PCR reaction during early &
exponential phase where the first significant increase
in the amount of PCR product correlates to the initial
amount of target template.
Applications of qPCR
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Gene Expression Profiling Analysis
Microarray Validation
miRNA Expression Profiling Analysis
Gene Regulation ‐‐‐ Genetic & Epigenetic
SNP Genotyping & allelic discrimination
Somatic Mutation Analysis
Copy Number Detection/Variation Analysis
Pathogen Detection
Viral Quantification
Considerations
• Isolation of mRNA from total RNA
(oligo dT) or random hexamer primers
• Choice of primers
• Amplicon size and GC content
Primers used for mRNA synthesis
SYBR-based Quantitative PCR
Amplification plot and
dissociation plot
Terminologies used in data analysis
Melting curve analysis
Real time qPCR analysis
• CT values = cycle number
•
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at which detectable signal is
achieved
Lower CT= Larger amount of starting material in the sample
Higher CT= Lower amount of starting material (template) in
the sample
Two basic methods of qPCR analysis
– Absolute quantification
– Relative quantification
Absolute quantification
Linear regression in quantification
Applications
• Viral load determination
• Gene copy number determination
Relative quantification
• To compare levels of gene expression
between mutants and wild type, treated
and untreated samples and in between
different organs/tissues.
Relative quantification
Relative quantification
Relative quantification
Plate set up
Data output
References
• http://sabiosciences.com/manuals/Intro
toqPCR.pdf
• http://relative.gene-quantification.info/
• http://www.genomics.agilent.com/
• http://www.protocol-online.org/
• http://www.qiagen.com/us/products/
• https://www.promega.com/products/