TRIZOL FLY RNA Extraction Protocol Nancy Linford 1/15/2010 Reagents 1. 1ml Trizol (NOT Trizol LS) per 100mg tissue (10-50 flies). Trizol is very caustic and requires specific disposal procedures. Please become aware of these before starting. All Trizol must be contained in the fume hood. 2. Chloroform 3. Isopropyl alcohol 4. Cold 70% Ethanol (in rnase free H20) 5. Rnase Free H20 6. Rnase inhibitor (100U/ul) RNA Isolation 1. Add up to 1ml Trizol to ribolyzer tubes. 2. Transfer flies IMMEDIATELY from -80 (or dry ice) to Ribolyzer tube. 3. Homogenize in Ribolyser at 6.5 (max) for 8s. 4. Leave tubes for 5min at RT. 5. Transfer to 1.5ml tube during RT incubation. 6. Add 1/5 V Chloroform and vortex for 15sec. 7. Incubate at RT for 3min. 8. Centrifuge at 12,000xg for 15min (4C). 9. Remove the aqueous upper layer to new tube, avoiding the interphase. 10. Add ½ V Isopropyl alcohol 11. Precipitate @RT 10 min. 12. Centrifuge at 12,000 for 10min (4C). 13. Wash with 1V 70% EtOH (cold, made with RNASE-free water) a. Make sure to sufficiently disrupt the pellet. Vortex 14. Spin at 8000 for 5min (4C). 15. Remove EtOH as completely as possible. a. Pulse spin after getting most out to bring rest to bottom and remove. 16. Air dry briefly and resuspend in 20ul RNAse free H20 + 0.5ul RNASE inhib. a. Do not resuspend by vigorous pipetting. Let the tubes stand at RT for a few minutes then flick to mix and briefly spin. 17. Heat at 55C for 60sec to help resuspension only if necessary. 18. Measure concentration on the Nanodrop & dilute all samples to the same concentration (0.1-1ug/ul) 19. (Optional) Run 2ug on bioanalyzer to determine the quality. <DATE> cDNA reaction (Applied Biosystems high capacity cDNA kit (P/N 4374966). See Applied Biosystems website for more information. Info in yellow should be filled inyou’re your experiment. http://www3.appliedbiosystems.com/cms/groups/mcb_support/documents/generaldocuments/cms_042557.pdf 1. Prepare all reagents on ice. # of samples 29 1ug RNA ul per rxn m. mix +RT m. mix -RT sample 10 10x Buffer 2 69.6 DNTP 0.8 27.84 Random primers 2 69.6 RT 1 34.8 2. H20 4.2 146.16 3. 4. Add 10ul of master mix to 10ul of sample in 0.2ml tubes. 5. In thermocycler: 10’ @ 25deg. 120’ @37 deg. 5” @85 deg. Store @-20deg RNA prep date: 4/17/09. sample ng/ul ul for h20 to Genes # name RNA 1ug 10ul to test notes 1 Name1 715.4 1.4 8.6 Gene1 2 Name2 472.8 2.1 7.9 Gene2 Etc… Goal: Real time PCR (using ABI Sybr green master mix) <DATE> 1. Use 8 well strip tubes or optical PCR plate with optical caps or optical film # of samples 50 ul per rxn m. mix cDNA 2 F primer (5uM) 2 120 R primer (5uM) 2 120 2x Sybr master mix 12.5 750 2. H2O 6.5 390 3. Spin down. 4. Run on ABI Step One Plus using sybr green default program with a melt step at the end. 5. Optimize dilution of cDNA for Cts of ~20-30. Include a std curve to ensure linearity and to calculate efficiency. 1 2 3 4 5 6 7 8 9 10 11 12 A B C D E F G H Notes: Blue=gene1, yellow=gene2. You should always compare a gene of interest to a reference gene.
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