2/17/2016 RAK
RNA Extraction – RT-PCR (modified from Lindy and Mei)
TC-Transfection
Grow cells to 50-60% confluence.
Transfect DNA using the FuGENE6 protocol.
(for p100 plates use 10ug total DNA, for p150 plates use 20ug DNA). For HFKs use 1:3
DNA:FuGENE ratio, for 293T cells use 1:4.
Samples:
FuGENE is added in a 3:1 to DNA (10 ul DNA at 1mg/ml conc with 30 ul FuGENE)
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Dilute FuGENE in serum free media (30 ul FuGENE in 270 ul media for each
transfection). Invert x3 and let rest for 5 min at RT.
Add 300 ul FuGENE/media dropwise to DNA to be transfected. Flick to mix and let rest
for 15 min at RT.
Add DNA/FuGENE mix dropwise to cells. Swirl plate to mix well.
Total RNA Extraction (Usual recovery is at least 200ug for a p150 plate)
Lyse cells at 24 hours to collect total RNA.
Wash cells once with cold PBS.
Lyse cells with 1-3ml Trizol for one confluent p100 plate; use 2-4 ml for one confluent
p150 plate.
Scrape with a spatula and transfer to a 15ml conical tube. (Lysate can be frozen at -80 degrees.)
Add 200ul CHCl3 per 1ml of Trizol. Vortex.
Spin for 30 minutes at 4 degrees at 3500 rpm (can do up to 5K) using tabletop eppy
centrifuge.
Transfer upper phase, with the RNA, into a 14ml falcon tube.
Precipitate RNA with 500ul isopropanol per ml of Trizol and 5ul glycogen azure.
Leave at -20 degrees for 1 hour or 10 min on dry ice. (Can leave overnight at -20 degrees.)
Spin at 9500 rpm for 20 minutes at 4 degrees in the sorvall (use rubber tube liners).
Wash with 5ml 75% EtOH (ice cold).
Spin at 7000 rpm for 5 minutes at 4 degrees.
Dry the pellet upside down in the hood.
Resuspend the RNA pellet with 100-200ul DEPC dH2O.
Heat at 65 degrees for 5 minutes.
Transfer to an eppy. OD to get quality and quantity of RNA (2ul in 500 DEPC dH2O).
Save samples at -80 degrees.
2/17/2016 RAK
Purify total RNA Samples
Use Rneasy kit (QIAGEN) to clean up RNA. OD to get quality and quantity of RNA.
Sample
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Concentration
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1ug total RNA
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RT Reaction (make DNA copy of RNA templates) (Have one control with no RNA
template)
Sample#
RNA (1ug total)
Random Hex primer (150ng/ul)
10mM dNTPs
Nuc. free dH2O (to total 10.5ul)
1
2
3
4
5
6
7
8
3
2.5
3
2.5
3
2.5
3
2.5
3
2.5
3
2.5
3
2.5
3
2.5
Master Mix
5x First strand buffer
RNAsin
0.1M DTT
dH2O
Total
9x
45
9
18
49.5
121.5
1x
5
1
2
5.5
13.5
Heat at 68 degrees for 5 minutes
Cool on ice for one minute
Add 13.5 to each sample. Total vol is 25ul per sample.
Mix gently and spin down briefly.
Add 1ul Superscript II RT to each tube. Mix gently.
Incubate at 25 degrees for 10 minutes, and then 42 degrees for 50 minutes.
Heat inactivate at 68 degrees for 15 minutes. May store at -20 degrees or use in RT-PCR
or real time PCR.
Reagents Used:
Trizol (Invitrogen, 15596-018)
Nuclease Free dH2O (Promega, P119C)
RNasin (Promega, N2111)
Random Primer (Invitrogen, 48190-011)
10mM dNTPs (Invitrogen, 18427-013)
Superscript II RT (Invitrogen, 18064-014), with 1st strand buffer and 0.1M DTT