Real Time PCR From Cell Culture: Difference between revisions

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__NOTOC__

==Real Time qPCR==

===Materials===

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*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~~)

*Primers (Dilute to 0.4 uM mixture of fwd and rev. From 100 uM stocks this is 4uL Forward Primer, 4 uL Reverse Primer and 992 uL Water)

*Generate primers using http://pga.mgh.harvard.edu/primerbank/index.html

==Protocol==

===RNA Extraction===

#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).

#Use RNEasy kit with Qiashredder. see [[Harvesting RNA from Cells grown in monolayer]] or [[Preparation_of_RNA_Samples_from_Mouse_Tissues]]. For a 12 well plate, use 350 uL of RLT (add 10 uL B-ME per 1 mL of RLT buffer).

#Scrape cells and pass through Qiashredder column. Do the optional DNAse step at step 5 using the RNAse free DNAse from Qiagen.

#Store at -20 until RT reaction

===RT-PCR Reaction===

#~~Use~~ 8 uL of RNA ~~per~~ RT ~~reaction~~.

#Book PCR Machine for 2h

#~~Use Superscript~~ RT~~-PCR kit from Invitrogen, following manufacturers instructions~~

#Thaw, mix and quickly spin RNA, dNTP mix, random hexamers, 10X RT buffer, 25 mM MgCl2 water. All reagents are in the small red invitrogen box

#Store cDNA at -20 until use

#Combine the following in a PCR tube:

~~#Typically dilute 5X in water (adjust for higher or lower expressing transcripts)~~

##8 uL RNA

##1 uL dNTP mix

##1 uL Random Hexamers

#Put in PCR machine and run program '''RT 65C''' (takes 5 min)

#In a separate tube add in the following order (for 10 reactions):

##20 uL 10X RT buffer

##40 uL 25 mM MgCl2

##20 uL 0.1M DTT

##10 uL RNAse Out

#Add 9 uL of the reaction mix to each primer/RNA mixture from previous step, mix and quickly centrifuge

#Sit on the bench for 2 min

#Add 1 uL SuperScript II RT to each tube

#Put in PCR machine and run program '''RT Reaction'''. After run (75 min) place on ice.

#Add 1 uL RNase H and put in PCR machine and run program '''RT 37'''. Takes 20 min.

#Store cDNA at -20 until use.

===Plate Preparation===

#Book 3h on qPCR machine at http://felix2.lsi.umich.edu/ORS~~/index.php?UID=2fd952b00de25~~

#Book 3h on qPCR machine at http://felix2.lsi.umich.edu/ORS, username '''and''' password is davebrid

~~#Prepare primer mix from 5 uM primer pair stocks by adding 0.4 uL primers + 4.6 uL water per reaction~~

#Get 96 or 384 well block and keep on rack. Do not touch bottom of plate.

#Get 96 well block and keep on rack. Do not touch bottom of plate.

#Add 5 uL template per well. If using a 384 well plate use 2.5uL

#Add 5 uL template per well.

#Add 5 uL primer per well. If using a 384 well plate use 2.5uL

#Add 5 uL primer per well

#Add 10 uL per row into 8 wells of a PCR strip plus an extra 10-20 uL

#Using a multichannel pipettor, add 10 uL Master mix to each well

#Using a multichannel pipettor, add 10 uL Master mix to each well. If using a 384 well plate use 5uL

#Start qPCR Machine using the machine in the [[qPCR - Lin Lab|Lin Lab]] or the [[qPRC - Saltiel Lab|Saltiel Lab]] machine

==Calculations==

see http://www.ncbi.nlm.nih.gov/pmc/articles/PMC55695 for considerations on calculations

==References (Saltiel Lab)==

~~<pubmed>~~18829989~~</pubmed>~~

PMID 18829989

~~<pubmed>~~17008399~~</pubmed>~~

PMID 17008399

~~<pubmed>~~17200717~~</pubmed>~~

PMID 17200717

~~<pubmed>~~17192460~~</pubmed>~~

PMID 17192460

~~<pubmed>~~16926380~~</pubmed>~~

PMID 16926380

[[Category:Transcription]]

[[Category:Expression]]

[[Category:RNA]]

[[Category:qPCR]]

@@ Line 1: / Line 1: @@
+__NOTOC__
 ==Real Time qPCR==
 ===Materials===
@@ Line 6: / Line 7: @@
 *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)
+*Primers (Dilute to 0.4 uM mixture of fwd and rev.  From 100 uM stocks this is 4uL Forward Primer, 4 uL Reverse Primer and 992 uL Water)
 *Generate primers using http://pga.mgh.harvard.edu/primerbank/index.html
 ==Protocol==
 ===RNA Extraction===
-#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).
+#Use RNEasy kit with Qiashredder.  see [[Harvesting RNA from Cells grown in monolayer]] or [[Preparation_of_RNA_Samples_from_Mouse_Tissues]].  For a 12 well plate, use 350 uL of RLT (add 10 uL B-ME per 1 mL of RLT buffer).
 #Scrape cells and pass through Qiashredder column.  Do the optional DNAse step at step 5 using the RNAse free DNAse from Qiagen.
 #Store at -20 until RT reaction
 ===RT-PCR Reaction===
-#Use 8 uL of RNA per RT reaction.
+#Book PCR Machine for 2h
-#Use Superscript RT-PCR kit from Invitrogen, following manufacturers instructions
+#Thaw, mix and quickly spin RNA, dNTP mix, random hexamers, 10X RT buffer, 25 mM MgCl2 water.  All reagents are in the small red invitrogen box
-#Store cDNA at -20 until use
+#Combine the following in a PCR tube:
-#Typically dilute 5X in water (adjust for higher or lower expressing transcripts)
+##8 uL RNA
+##1 uL dNTP mix
+##1 uL Random Hexamers
+#Put in PCR machine and run program '''RT 65C''' (takes 5 min)
+#In a separate tube add in the following order (for 10 reactions):
+##20 uL 10X RT buffer
+##40 uL 25 mM MgCl2
+##20 uL 0.1M DTT
+##10 uL RNAse Out
+#Add 9 uL of the reaction mix to each primer/RNA mixture from previous step, mix and quickly centrifuge
+#Sit on the bench for 2 min
+#Add 1 uL SuperScript II RT to each tube
+#Put in PCR machine and run program '''RT Reaction'''.  After run (75 min) place on ice.
+#Add 1 uL RNase H and put in PCR machine and run program '''RT 37'''.  Takes 20 min.
+#Store cDNA at -20 until use.
 ===Plate Preparation===
-#Book 3h on qPCR machine at http://felix2.lsi.umich.edu/ORS/index.php?UID=2fd952b00de25
+#Book 3h on qPCR machine at http://felix2.lsi.umich.edu/ORS, username '''and''' password is davebrid
-#Prepare primer mix from 5 uM primer pair stocks by adding 0.4 uL primers + 4.6 uL water per reaction
+#Get 96 or 384 well block and keep on rack.  Do not touch bottom of plate.
-#Get 96 well block and keep on rack.  Do not touch bottom of plate.
+#Add 5 uL template per well.  If using a 384 well plate use 2.5uL
-#Add 5 uL template per well.
+#Add 5 uL primer per well.  If using a 384 well plate use 2.5uL
-#Add 5 uL primer per well
 #Add 10 uL per row into 8 wells of a PCR strip plus an extra 10-20 uL
-#Using a multichannel pipettor, add 10 uL Master mix to each well
+#Using a multichannel pipettor, add 10 uL Master mix to each well.  If using a 384 well plate use 5uL
+#Start qPCR Machine using the machine in the [[qPCR - Lin Lab|Lin Lab]] or the [[qPRC - Saltiel Lab|Saltiel Lab]] machine
+==Calculations==
+see http://www.ncbi.nlm.nih.gov/pmc/articles/PMC55695 for considerations on calculations
 ==References (Saltiel Lab)==
-<pubmed>18829989</pubmed>
+PMID 18829989
-<pubmed>17008399</pubmed>
+PMID 17008399
-<pubmed>17200717</pubmed>
+PMID 17200717
-<pubmed>17192460</pubmed>
+PMID 17192460
-<pubmed>16926380</pubmed>
+PMID 16926380
+[[Category:Transcription]]
+[[Category:Expression]]
+[[Category:RNA]]
+[[Category:qPCR]]