Validation of Hits from an sirna Library Screen using Real-Time qpcr. Gregory L. Shipley The University of Texas Health Science Center- Houston

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1 Validation of Hits from an sirna Library Screen using Real-Time qpcr Gregory L. Shipley The University of Texas Health Science Center- Houston

2 sirnas and Gene Silencing Small interfering RNAs or sirnas are short, double stranded molecules that may contain modifications on one strand sirnas target specific sequences within the message Following transfection into the cell, the sirna may or may not be processed by Dicer, depending on length. The base pair sirna is incorporated into a ribonuclear protein complex that becomes the RISC complex (RNA-induced silencing complex) following the addition of other proteins Within the RISC, the sirna is unwound in an ATP dependent manner Formation of an mrna/sirna duplex leads to transcript degradation and recycling of the RISC

3 sirna Library Screening Invitrogen Kinome Set A Set B Set C

4 The Infrastructure of Screening

5 The Infrastructure of Screening

6 Basic Procedure for an sirna Library Screen Printing of library and +/- control sirnas into 96- or 384-well plates Reverse transfection of sirna into cells Growth period +/- the addition of drugs or other activators in hours post-plating Assay for sirna knockdown effect using an assay of interest in hours - biochemical- or imaging-based readouts cell viability apoptosis nuclear translocation, etc.

7 A Quick Measure of Transfection Efficiency Must have the correct reverse transfection reagent for the experimental cell type How to determine this quickly? Use a scrambled sirna with a conjugated fluorescent dye Transfect using many reverse transfection reagents and the fluorescently tagged sirna Monitor under a manual or automated fluorescent

8 A Quick Measure of Transfection Efficiency The same reverse transfection reagent will not necessarily be optimal for all cell lines Control - HeLa Vendor 2 Vendor 1 Roche X-treme Gene

9 Goals for using Real-Time qpcr for Secondary Screening of sirna Hits Make the sirna knockdown validation step as amenable to automation as possible Use one set of cells to repeat the original assay results and a companion set to monitor knockdown by real-time qpcr Stabilize the RNA in one set of cells while assaying the replicate companion set Make cdna directly from cell lysates to avoid the time, cost and potential effect on the final data set due to RNA isolation

10 Stablization of RNA in situ Looked at how stable RNA would be following formalin fixation Not as bad as you might think, not as good as you would like Final answer is RNALater Cells are stable at 4 C for weeks as advertised

11 Stable Lysates from RNALater Preserved Cells Tested 3 different products- Sidestep (Stratagene), Cells Direct (Invitrogen) and Cells to cdna (Ambion) All produced usable lysates that gave comparable results for the target transcript but not for DNA contamination Chose the Cells to cdna reagent for ease of use and we thought the best results for our application

12 Comparison of Different Lysis Reagents Cells Direct- 20 µl reagent/well Cells to cdna- 20 µl reagent/well Sidestep- 20 µl reagent/well

13 Optical and Real-Time qpcr Results HeLa AR-YFP stable cell line AR618 Control + DHT 2H AR619 Control +mgc sirna & DHT 2H AR620

14 Optical Data Analysis

15 Optical and Real-Time qpcr Results HeLa AR-YFP stable cell line Procedure- real-time qpcr One set of cells washed 1X in cold PBS and preserved with RNALater - 30 µl/well (96-well plate, 24 hours, 4 C) Wash 2X in cold PBS & add 30 µl/well Cells to cdna reagent, pipet up/down and transfer to PCR plate 75 C- 10 minutes DNase I treat 37 C-15 min, 75 C- 5 min Assay for β-actin and AR levels via real-time qpcr

16 β-actin sirna Knockdown of AR using Invitrogen sirnas Androgen Receptor

17 Summary Goal 1: make the procedure as automation friendly as possible- all but the 75 C heating step Goal 2: preservation of RNA in situ during the validation step- done by using RNALater Goal 3: synthesize cdna directly from cell lysates, works very well using Cells to cdna