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. 18 th ) 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

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 /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  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 C T values = cycle number at which detectable signal is achieved Lower C T= Larger amount of starting material in the sample Higher C T= 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

To compare levels of gene expression between mutants and wild type, treated and untreated samples and in between different organs/tissues. Relative quantification

Plate set up

Data output

References toqPCR.pdf toqPCR.pdf