Difference between revisions of "Real Time PCR From Cell Culture"

Revision as of 20:11, 26 May 2009

Real Time qPCR

Materials

• cDNA for templates
• Qiashredder and RNEasy kits from Qiagen
• Superscript Kit from Invitrogen
• SyberGreen PCR Master Mix Applied Biosystems
• 96 well qPCR plate
• Primers (Dilute to 5 uM mixture of fwd and rev and then make a working solution of 0.4 uL + 4.6 uL water per reaction)
• Generate primers using http://pga.mgh.harvard.edu/primerbank/index.html

Protocol

RNA Extraction

1. Use RNEasy kit with Qiashredder. see Harvesting RNA from Cells grown in monolayer. For a 12 well plate, use 350 uL of RLT (add 10 uL B-ME per 1 mL of RLT buffer).
2. Scrape cells and pass through Qiashredder column. Do the optional DNAse step at step 5 using the RNAse free DNAse from Qiagen.
3. Store at -20 until RT reaction

RT-PCR Reaction

1. Use 8 uL of RNA per RT reaction.
2. Use Superscript RT-PCR kit from Invitrogen, following manufacturers instructions
3. Store cDNA at -20 until use
4. Typically dilute 5X in water (adjust for higher or lower expressing transcripts)

Plate Preparation

1. Book 3h on qPCR machine at http://felix2.lsi.umich.edu/ORS/index.php?UID=2fd952b00de25
2. Prepare primer mix from 5 uM primer pair stocks by adding 0.4 uL primers + 4.6 uL water per reaction
3. Get 96 well block and keep on rack. Do not touch bottom of plate.
4. Add 5 uL template per well.
5. Add 5 uL primer per well
6. Add 10 uL per row into 8 wells of a PCR strip plus an extra 10-20 uL
7. Using a multichannel pipettor, add 10 uL Master mix to each well

References (Saltiel Lab)

<pubmed>18829989</pubmed> <pubmed>17008399</pubmed> <pubmed>17200717</pubmed> <pubmed>17192460</pubmed> <pubmed>16926380</pubmed>