Presentation on theme: "RNA Lab (Isolation, quantification and qPCR analysis) MCB7300."— Presentation transcript:
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.
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.
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.
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