Preparation of RNA for Microarray Analysis. Protocol Outline

Transcription

1 J. Perez / 06 Protocol Outline Day 0 Day 1 Sort and RNA extraction with Trizol Round 1: 1 st strand cdna synthesis Round 1: 2 nd strand cdna synthesis Round 1: cdna purification Overnight Day 1/Day 2 Round 1: arna synthesis (IVT) Day 2 Day 3 Round 1: arna purification, precipitation, and quantification Round 2: 1 st strand cdna synthesis Round 2: 2 nd strand cdna synthesis Round 2: cdna purification Round 2: arna synthesis with biotin labeling Round 2: Purification of labeled arna Materials: - MessageAmp arna kit (Ambion Cat # 1750) One round of amplification for 20 samples, or two rounds for 10 - BioArray HighYield RNA Transcript Labeling Kit (T7) (Enzo No for 10 samples or for 20 samples) - RNeasy RNA purification spin column kit (Qiagen 74104) RNase ZAP Large bore (hole) 1ml tips 0.2 ml thin walled PCR tubes 1.5 ml eppendorf tubes Trizol Chloroform Glycoblue Isopropanol DEPC water 100% and 70% ethanol (RT and cold) RNase Inhibitor (Promega) 2M NaOAC (sodium acetate) TE β-mercaptoethanol 1

2 Day 0: Sort and RNA extraction with Trizol 1 Sort cells into 1.5 ml epp. tubes coated in Fetal Calf Serum (FCS) (or PBS or medium + FCS) 2 Centrifuge 3 4,000 g, 4 C (make sure to see a pellet, can try 30 full speed) 3 Remove supernatant from each sample with a pipette tip Leave behind a small amt of liquid so as not to disturb the pellet 4 Add 500 μl Trizol to each sample (mix by pipetting) 5 Incubate 5 RT (option: Freeze in Trizol at least C ) 6 Add 100 μl Chloroform to each sample (work in hood) Shake vigorously for approx 15 sec 7 Incubate 2-3 RT 8 Centrifuge 15 12,000 g, 4 C 9 Transfer aqueous phase (approx μl) CAREFULLY to a fresh tube 1 (from now on, keep on ice in between steps) 10 Add 1μl Glycoblue to each sample (flick) 11 Add 250μl isopropanol (flick) Incubate 10 RT (or C) 12 Centrifuge 15 12,000 g, 4 C Remove supernatant with a pipette tip 13 Wash with 500μl 75% ethanol (flick) 14 Centrifuge 5 7,400 g, 4 C Pipette off as much ethanol as possible, air dry 15 Resuspend each RNA pellet in 15 μl Nuclease-free water RT for 1-2 min, then flick to mix -80 C until ready to proceed) Day 1: Round 1: 1 st strand cdna synthesis 1 Transfer 11 µl of RNA to 0.2 ml thin walled PCR tubes 2 Add 1 µl of T7 Oligo(dT) RT (flick, spin) 3 Incubate 10 70ºC 2 (spin, ice) 4 Assemble master RT (flick, spin, ice) (be conservative when assembling this mix) 2 µl 10x First Strand Buffer 3 1 µl RNase Inhibitor (from kit) 4 µl dntp Mix 1 µl Reverse Transcriptase 5 Add 8 µl mix to each sample (flick, spin) 1 Leave behind a small amount of clear aqueous phase; do NOT pick up any pink phenol-chloroform phase; use pipette tips with a larger hole to prevent this from happening. 2 Use PCR machines for incubations Set lid to tracking 5ºC: Setup lid tracking 5ºC. Select 3 10x First Strand Buffer might precipitate warm to RT to dissolve. 2

3 6 Incubate 2 42ºC (spin, ice) Day 1: Round 1: 2 nd strand cdna synthesis 1 Assemble master mix on ice (gently mix, spin) 1 (again, be conservative) 63 µl Nuclease-free water 10 µl 10x Second Strand Buffer 2 4 µl dntp mix 2 µl DNA Polymerase 1 µl RNase H 2 Add 80 µl mix to each sample (flick, spin) 3 Incubate 2 16ºC 3 (spin, ice or 20ºC) Day 1: Round 1: cdna purification For new kits add 11.2 ml 100% ethanol to cdna Wash Buffer and 20 ml to arna Wash Buffer 1 Preheat Nuclease-free water 50ºC 4 2 Put cdna filter cartridges into 2 ml wash tubes 3 Equilibrate filter cartridge with 50 µl cdna Binding Buffer 5 RT for 5 min (do not spin) 4 Transfer cdna samples to 1.5 ml epp. tubes 5 Add 250 µl cdna Binding Buffer to each sample (mix by pipetting) Apply mixture to corresponding filter cartridge 6 Centrifuge 1 10,000 rpm 6 Discard flow-through and replace filter in tube 7 Apply 500 µl cdna Wash Buffer to each filter cartridge 8 Centrifuge 10,000 rpm Discard flow-through; Centrifuge again 1 10,000 rpm 9 Transfer cdna filter cartridges to cdna elution tubes 10 Elute cdna by applying 10 µl preheated Nuclease-free water to filter 11 Centrifuge ,000 rpm 12 Elute again with 10 µl preheated Nuclease-free water 13 Centrifuge again ,000 rpm 1 Do not use p1000 for measuring out these amounts measurements need to be as accurate as possible since pippeting error is not taken into consideration here. 2 10x Second Strand Buffer might precipitate warm to RT to dissolve. 3 Use PCR machines for incubations Set lid to tracking 5ºC: Setup lid tracking 5ºC. Select 4 Keep Nuclease-free water at 50ºC as much as possible between elutions. 5 cdna Binding Buffer might precipitate warm to 37ºC to dissolve then cool to RT. 6 Centrifugation should be done at room temp. 3

4 Transfer no more than 18 µl of cdna to 0.2 ml PCR tubes (ice or 20ºC) Overnight Day 1/Day 2: Round 1: arna synthesis (IVT) 1 Assemble master RT (gently mix, spin) 1 (no need to be conservative here) 4 µl T7 ATP Solution 4 µl T7 CTP Solution 4 µl T7 GTP Solution 4 µl T7 UTP Solution 4 µl T7 10x Reaction Buffer 2 4 µl T7 Enzyme Mix 1 µl RNaseIn (Promega) 2 Add 25 µl mix to each cdna sample (flick, spin) 3 Incubate 6 14 hr (usually 14 hr, but no 37ºC 3 4 Add 2 µl DNaseI to each sample (flick, spin) 5 Incubate 30 37ºC 6 Transfer samples to 1.5 ml epp. tubes 7 Add 60 µl arna Elution Solution to each sample (mix by pipetting, spin) Day 2: Round 1: arna purification, precipitation, and quantification Purification: 1 Preheat Nuclease-free water 50ºC 4 2 Put arna filter cartridges into arna collection tubes 3 Add 350 µl arna Binding Buffer to each sample (mix by pipetting, spin) 4 Add 250 µl 100% ethanol to one sample (mix by pipetting) Immediately apply to corresponding filter cartridge Repeat for all samples 5 Centrifuge 1 10,000 rpm 5 Discard flow-through and replace filter in tube 6 Apply 650 µl arna Wash Buffer to each filter cartridge 7 Centrifuge 1 10,000 rpm Discard flow-through; Centrifuge again 1 10,000 rpm 1 Since Round 2: arna synthesis with biotin labeling requires a different kit, feel free to scale up this master mix to account for pipetting error. 2 T7 10x Reaction Buffer might precipitate warm to RT to dissolve. 3 Use PCR machines for incubations Set lid to tracking 5ºC: Setup lid tracking 5ºC. Select 4 Keep Nuclease-free water at 50ºC as much as possible between elutions. 5 Centrifugation should be done at room temp. 4

5 8 Transfer arna filter cartridges to arna collection tubes 9 Elute arna by applying 50 µl preheated Nuclease-free water to filter 10 Centrifuge 2 10,000 rpm 1 11 Elute again with 100 µl preheated Nuclease-free water 12 Centrifuge 2 10,000 rpm Precipitation: 13 Precipitate arna by directly adding to each sample 22.5 µl 2M NaOAc (vortex) 0.5 µl Glycoblue (vortex, or flick, spin) 450 µl cold 100% ethanol (vortex, spin, ice) 14 Incubate 1 -80ºC (use freezer) 15 Centrifuge 20 13,200 rpm 16 Discard supernatant; Wash with 500 µl cold 70% ethanol (vortex) Centrifuge 5 13,200 rpm 17 Wash again with 500 µl cold 70% ethanol (vortex) Centrifuge 5 13,200 rpm 18 Remove remaining ethanol with pipette tip Cover loosely with saran wrap and air dry briefly 19 Resuspend arna in 15 µl Nuclease-free water (ice or 80ºC) Quantification: 20 Dilute 1 µl arna in 99 µl TE for quantification 2 21 Measure OD 260 and OD 260 /OD 280 with Nucleic Acids setting Conversion: µg/ml = ng/µl = OD 260 x dilution factor x 40 µg/ml Purity of sample = OD 260 /OD Move on to 2 nd Round or freeze overnight 80ºC 1 Centrifugation should be done at room temp. 2 Always OD RNA and DNA in TE, not water. Remember to turn UV light on ahead of time. 5

6 Day 2: Round 2: 1 st strand cdna synthesis 1 Add 2 µg arna to 0.2 ml PCR tubes 2 Add Nuclease-free water up to 10 µl final volume 3 Add 2 µl 2 nd Round Primer (flick, spin) 4 Incubate 10 70ºC 1 (spin, ice) 5 Assemble master RT (flick, spin) (be conservative when assembling this mix) 2 µl 10x 1 st Strand Buffer 2 1 µl RNase Inhibitor (from kit) 4 µl dntp mix 6 Add 7 µl master mix to each sample (flick, spin) 7 Incubate 10 25ºC (spin) 8 Add 1 µl Reverse Transcriptase to each sample (flick, spin) 9 Incubate 2 42ºC (spin) 10 Add 1 ul RNase H to each sample (flick, spin) 11 Incubate 30 37ºC (spin) Day 2: Round 2: 2 nd strand cdna synthesis 1 Add 5 µl T7 Oligo(dT) Primer to each sample (flick, spin) 2 Incubate 10 70ºC (spin, ice) 3 Assemble master mix (gently mix, spin) 3 (be conservative when assembling this mix) 58 µl Nuclease-free water 10 µl 10x 2 nd Strand Buffer 4 4 µl dntp mix 2 µl DNA Polymerase 4 Add 74 µl of master mix to each 26 µl sample (gently mix, spin) 5 Incubate 2 16ºC (spin) 1 Use PCR machines for incubations Set lid to tracking 5ºC: Setup lid tracking 5ºC. Select 2 10x First Strand Buffer might precipitate warm to RT to dissolve. 3 Do not use p1000 for measuring out these amounts measurements need to be as accurate as possible since pippeting error is not taken into consideration here. 4 10x Second Strand Buffer might precipitate warm to RT to dissolve. 6

7 Day 2: Round 2: cdna purification 1 Preheat Nuclease-free water 50ºC 1 2 Put cdna filter cartridges into 2 ml wash tubes 3 Equilibrate filter cartridge with 50 µl cdna Binding Buffer 2 RT for 5 min (do not spin) 4 Transfer cdna samples to 1.5 ml epp. tubes 5 Add 250 µl cdna Binding Buffer to each sample (mix by pipetting) Apply mixture to corresponding filter cartridge 6 Centrifuge 1 10,000 rpm 3 Discard flow-through and replace filter in tube 7 Apply 500 µl cdna Wash Buffer to each filter cartridge 8 Centrifuge 10,000 rpm Discard flow-through; Centrifuge again 1 10,000 rpm 9 Transfer cdna filter cartridges to cdna elution tubes 10 Elute cdna by applying 10 µl preheated Nuclease-free water to filter 11 Centrifuge ,000 rpm 12 Elute again, this time with 16 µl preheated Nuclease-free water 13 Centrifuge again ,000 rpm Transfer 22 µl of cdna to 0.2 ml PCR tubes (ice or store at 20ºC until ready to proceed) Day 3: Round 2: arna synthesis with biotin labeling - Enzo BioArray HighYield RNA Transcript Labeling Kit (T7) 1 Assemble master RT (flick, spin) (be conservative when assembling this mix) 4 μl 10x Rxn Buffer (vial 1) 4 μl Biotin-labeled Nucleotides (vial 2) 4 μl 10x DTT (vial 3) 4 μl 10x RNase Inhibitor (vial 4) 2 μl 20x T7 RNA Polymerase (vial 5) 2 Add 18 µl of master mix to each 22 µl cdna sample (flick, spin) 3 Incubate 5 37 C 4 (mix gently every 45 min) 1 Keep Nuclease-free water at 50ºC as much as possible between elutions. 2 cdna Binding Buffer might precipitate warm to 37ºC to dissolve then cool to RT. 3 Centrifugation should be done at room temp. 4 Use PCR machines for incubations Set lid to tracking 5ºC: Setup lid tracking 5ºC. Select 7

8 Day 3: Round 2: Purification of labeled arna - Qiagen RNeasy RNA purification spin column kit Prepare RLT buffer fresh: 350 μl/sample needed, must add 10 μl β-me (β-mercaptoethanol aka 2-Mercaptoethanol) per 1 ml buffer just prior to use For new kits add 4 volumes of 100% ethanol to RPE buffer 1 Put RNeasy columns into 2 ml collection tubes 2 Add 60 μl Nuclease-free water to each RT; transfer to epp. tubes 3 Add 350 μl RLT to each sample (mix by pipetting) 4 Add 250 μl 100% ethanol (mix by pipetting) Apply the 700 μl of mixture onto appropriate RNeasy column 5 Centrifuge 15 10,000 rpm 1 Discard collection tube and flow-through; transfer column to fresh 2 ml collection tube 6 Add 500 μl RPE to each column 7 Centrifuge 15 10,000 rpm Discard flow-through 8 Apply 500 μl RPE again to each column 9 Centrifuge 2 rpm Discard flow-through; transfer column to epp. tube (with removed lid) for collection 10 Centrifuge again 1 full speed to remove leftover ethanol Transfer column to fresh 1.5 ml collection tube 11 Elute RNA by applying 30 μl Nuclease-free water 12 Centrifuge 1 10,000 rpm 13 Elute again with another 30 μl Nuclease free water 14 Centrifuge 1 10,000 rpm 15 Quantify 1 μl arna in 99 μl TE 2 16 Aliquot 15 μg of arna for chip analysis RNA should be at least 0.6 μg/μl or higher 1 Centrifugation should be done at room temp. 2 Always OD RNA and DNA in TE, not water. Remember to turn light on ahead of time. 8