Applied Biosystems 7900HT Fast Real-Time PCR System I. Real-time RT-PCR analysis of siRNA-induced knockdown in mammalian cells (Amit Berson, Mor Hanan and Dr. Mira Korner)

Experimental outline 1.We will use siRNA in order to knockdown the expression of two prototypic splicing factors, SC35 and ASF/SF2 2.Examine the expression of the two splicing factors in HeLa cell line after siRNA transfection using Real time RT-PCR 3.Furthermore, we will examine the expression of MKNK2 and caspase9 splice variants. The alternative splicing of these genes are known to be mediated by ASF/SF2.

Standard assays for RNA quantification Micro-arrays – allows the comparison of many thousands of transcripts between different samples. However, results must be validated with conventional RT-PCR Gel based RT-PCR. RT to generate cDNA, PCR amplification for various number of cycles and gel electrophoresis. The use of this method is rapidly decreasing with the increasing popularity of real-time PCR. This method is both time-consuming and to a great extent less accurate than real-time PCR Northern blot – Direct visualization of the RNA with radioactive probe.

Standard assays for RNA quantification In-situ hybridization – the only method that detects RNA molecules in their natural environment of the cell. This method is less accurate for quantification but has the enormous advantage of examining RNA levels in cell-type specific / intracellular localization specific manner And more…. RNase protection etc. Real-time PCR – allows monitoring of product accumulation in real-time during the PCR process. This method has several major advantages: accurate quantification, ability to distinguish between and primer-dimer or a non-specific product, and easy handling and less time consuming

Basics of real-time RT-PCR Total RNA Reverse transcription using random or poly dT primers cDNA Polymerase chain reaction (PCR) using specific primers Amplified product

Real-time PCR assays

Advantages: High specificity (no primer dimers) Disadvantages: Requires probe design and synthesis or the use of commercially available ready made and tested probes More expensive Real-time PCR assays Basic chemistry suitable for all amplicons - only primer design is needed Lower specificity

fluorescence cycle Amplification curve Threshold cycle (C T ) In the exponential phase, DNA is amplified by approximately 2-fold in each cycle

temperature fluorescence Melting curve derivative temperature Cool down 95ºC 1 degree intervals

Primer design All primers must have similar Tm (Melting temp.) we use Tm of 59º ±1 Amplicons must be similar in length – we use approx. 80bp amplicons Primers are designed to anneal to an exon-exon junction or to flank an intron, to avoid amplification of genomic DNA Forward primer Reverse primer exon intron exon

Normalization: Reference genes Use of an appropriate standard to normalize results is essential as pipetting errors and differences in starting material amounts may lead to misinterpretation of the results What is an appropriate standard? Several genes are commonly used as reference genes. However, for each study a specific suitable reference gene, or a combination of several genes should be used. For example: β-actin 18S RNA GAPDH Etc. In addition, well to well variability is normalized using a passive reference fluorescent molecule - ROX

Controls Non template control : tests contamination of stock solutions -RT : tests carry over of genomic DNA

Absolute Vs. relative quantification Absolute quantification is performed using amplification of known amounts of RNA and plotting a standard curve of C T Vs. log of quantity Relative quantification allows comparison of mRNA levels between different samples without the need of absolute quantification

Thermal program: 50º 2.00 UNG… 95º to start the polymerase activity 95º 0.15 double strands disassociation 60º 1.00 annealing, elongation, and fluorescence detection X40 Melting curve

Serial dilutions In order to validate that changes in RNA concentration result in appropriate change in C T (threshold cycle) we perform real-time PCR on serial dilution of a sample 1:1 1:21:4… <<< From this data we can also deduce the primer efficiency: Eff = 10^(1/slope)

Primer dimer or non-specific amplicons effect can be minimized, BUT their amplification makes quantification inaccurate No primer dimerPrimer dimer Reading temp: 60º Reading temp: 80º