RNA EXTRACTION PROTOCOL 3/10/2016 (ON COLUMN DNase)
1) Concentrate worms from at least 2 plates per condition so the final yield increases. Following this protocol can give you at least 5ug RNA total if RNA is being purified from adult worms (or mixed populations) from worms coming from NAMM. The quality of the RNA obtained measured by A260/230 and A260/280 ratios if RNAeasy columns were used, is around 2.2.
2) Wash NGM or NAMM plates with M9 and pellet worms in Eppendorf tubes. Discard supernatant leaving about 100ul of M9. Do not store worms at -80C.
3) Add 1ml of Trizol to each sample. Rapidly thaw and freeze the eppendorfs containing Trizol in liquid nitrogen or -70 degrees C Isopropanol+Dry Ice bath at 5X. This ensures adequate worm lysis.
4) Vortex the eppendorfs at room temperature for 30 minutes.
5) Spin the worms at max speed at 4 degrees C for 5 minutes. Decant the clear supernatant to freshly autoclaved tubes without disturbing the pellet.
6) Add 200ul of chloroform (in the fume hood) and close the eppendorfs firmly. Vortex the eppendorfs for 10 minutes at room temperature. This allows for mixing of the Trizol and choloform layers. Spin the eppendorfs at max speed at 4C for 10 minutes to separate the aqueous and organic layers. Repeat this step once more with the aqueous layer.
7) Separate the colorless and transparent aqueous layer to fresh tubes. Add in this order 6ul of glycoblue, then either (500ul of isopropanol) OR (150ul of 3M sodium acetate pH 5.5 and 1 ml of absolute ethanol). Vortex briefly and place the eppendorfs at -20C at least 3h but not more than 5h, do not store overnight.
8) Spin the eppendorfs at max speed at 4C for 25 minutes.
9) After the spin you will see a soft blue precipitate. Discard the supernatant completely without disturbing the pellet. Add 100 microliters of 70% ethanol.
10) Spin the tube at max speed at 4C for 10 minutes.
11) Discard the supernatant and try to remove the alcohol without disturbing the pellet, usually by pipetting. Air-dry for 10 minutes.
12) Resuspend pellet in 100 microliters of RNAse free water.
13) From the RNA kit (we use QIAGEN RNeasy mini kit), 350ul of Buffer RW1 to an RNA column and centrifuge at 10k rpm for 15 seconds.
14) Add 350ul of Buffer RLT to the RNA in water and mix thoroughly.
15) Add 250ul of 100% ethanol to the RNA mixture and mix thoroughly. Add mixture to RNA column. Close gently and centrifuge for 15 seconds at 10k rpm.
16) Make solution of 70ul of water, 2ul DNAse, and 8ul of DNAse buffer and add to column. Incubate at 37C for 30 minutes.
17) Add 350 microliters of RW1 to column and spin for 15 sec @ 10k rpm.
18) Pipet 500ul of Buffer RPE onto spin column. Close lid and spin for 15 sec @ 10k rpm.
19) Add 500ul of 80% ethanol to spin column. Close the lid and spin for 2 minutes at 10k rpm.
20) Transfer column to new collection tube and spin for 5 min at full speed with the lid open to dry the membrane.
21) Elute RNA using 18ul HOT RNAse free water (5min, 55C), transfer the column to a new eppendorf and elute again with the same volume of water, so you can recover more RNA.
22) Nanodrop the RNA to determine concentration.
EPPENDORF DNASE DIGESTION
12) SAME AS PREVIOUS PROTOCOL STEP 12
13) For each RNA sample in 100 microliters of water, add 2 microliters of DNase and 10 microliters of DNase buffer. Incubate at 37 degrees C for at least 30 minutes.
14) After incubation, add 350 microliters of Buffer RLT to each RNA sample and mix well.
15) Add 250 microliters of 100% ethanol and mix well
16) Add total volume of RNA+ RLT + EtOH (~750 microliters) to RNA spin column, centrifuge at 10,000 RPM for 30 seconds
17) Throw away eluent from column, add 500 microliters of Buffer RPE to column, centrifuge at 10,000 RPM for 30 seconds.
18) Throw away eluent from column, add 500 microliters of Buffer RPE to column, centrifuge at 10,000 RPM for 2 minutes
19) Throw away eluent. Put column in new collection tube. Spin column for 1 minute at full speed of the centrifuge.
20) Put column into microcentrifuge tube. Add 14-25 microliters of RNase free water to column and let sit for at least 1 minute. Centrifuge for 2 minutes at max centrifuge speed to elute RNA in water.
22) Determine RNA concentration (Nanodrop or other mechanism)
23) Use cDNA synthesis kit to make cDNAs. Most kits use 10 microliters of RNA + water (I go for 100 ng of RNA per reaction but have gotten results from 20 ng) and 10 microliters of the reverse transcriptase reaction mix.
REAGENTS WE USE/YOU WILL NEED
TRIzol reagent- Ambion/Life Technologies. Can purchase in Biochem Stockroom I believe
GlycoBlue –Ambion. Not sure anyone on campus sells it
Turbo DNase – Ambion Not sure if it sold on campus
RNeasy kit- Qiagen. Sold in biochem stockroom. OR
Macherey-Nagel RNA cleanup kit. Columns sold in stockroom but I am not sure about buffers
We use the High Capacity cDNA Reverse Transcription Kits from Applied Biosystems. Can buy these at the Biochem Stockroom.