gDNA contamination in realtime PCR - (Sep/02/2007 )
Hi all
A few queries related with real time PCR-
*The Ct values in gDNA control varies from 31 to 24, is this a significant contamination in the exp? As far as I know it impossible to completely eliminate gDNA from RNA.how can I get rid off this contamination?I isolate total RNA by trizol n then purify it by Qiagen kit...I dont give DNaseI treatment since this reduced the A260/280 ratio significantly( I use fermantas Dnase I), they recommend inactivation of DNase by heating at 65 degree C, should i extract it with chloroform- isoamyl?
*In the replicate experiments how much variation in Ct value is permissible, the Ctvalues of same gene in 3 replicates are 24.9, 24 & 25.8....is this a great variation? Wat is the basic fact behind determination of range of Ct values for replicates?
*how much variation in Ct value( concerned gene of interest ) is considered to be significant? The average Ct value of 25.5( for treated) & 24.9 ( control) replicates can be regarded as significant change or not?
I follow delta delta Ct metod for analysis....
Thanx
bye
A few queries related with real time PCR-
*The Ct values in gDNA control varies from 31 to 24, is this a significant contamination in the exp? As far as I know it impossible to completely eliminate gDNA from RNA.how can I get rid off this contamination?I isolate total RNA by trizol n then purify it by Qiagen kit...I dont give DNaseI treatment since this reduced the A260/280 ratio significantly( I use fermantas Dnase I), they recommend inactivation of DNase by heating at 65 degree C, should i extract it with chloroform- isoamyl?
*In the replicate experiments how much variation in Ct value is permissible, the Ctvalues of same gene in 3 replicates are 24.9, 24 & 25.8....is this a great variation? Wat is the basic fact behind determination of range of Ct values for replicates?
*how much variation in Ct value( concerned gene of interest ) is considered to be significant? The average Ct value of 25.5( for treated) & 24.9 ( control) replicates can be regarded as significant change or not?
I follow delta delta Ct metod for analysis....
Thanx
bye
another possibility to eliminate gDNA amplification is to design your qPCR primers so
that one primer lies between to exons (intron spanning) so you only get an amplification product with mRNA because there the intron is spliced out
and genomic DNA would not be amplified
of course you have to have a gene with introns...
I'd recomend DNA free from Ambion, it works really well.
also, definitely- when posible- choose primers that expand and intron, is the best way to make sure.
also, definitely- when posible- choose primers that expand and intron, is the best way to make sure.
Removing the gDNA can be efficiently done by using RQ1 DNAse I from Promega, followed by inactivation with 2M Guanidine HCL and then phenol/cholorofrm/isoamyl Extraction and NaOACH/ETOH precipitation. The real trick to getting rid of all of the gDNA is to pipet the DNAseI into your sample, quickly centrifuge and then to remove the sample into a new Dnase/Ranse free tube. This makes sure that there is no gDNA that escapes DNAse I treatment.
Designing primers that are cross intron, (i.e. multi-exon) may or may not get rid of apparent gDNA contamination because of the presence of pseudogenes. Depending on the target sequence there may be a few (<10) to a lot (>50) potential pseudogenes that will be amplified with cross intron primers. It may be easier to remove this "contamination" with Taqman based probes than with SYBR green reactions.
Hopes this helps:
also, definitely- when posible- choose primers that expand and intron, is the best way to make sure.
Removing the gDNA can be efficiently done by using RQ1 DNAse I from Promega, followed by inactivation with 2M Guanidine HCL and then phenol/cholorofrm/isoamyl Extraction and NaOACH/ETOH precipitation. The real trick to getting rid of all of the gDNA is to pipet the DNAseI into your sample, quickly centrifuge and then to remove the sample into a new Dnase/Ranse free tube. This makes sure that there is no gDNA that escapes DNAse I treatment.
Designing primers that are cross intron, (i.e. multi-exon) may or may not get rid of apparent gDNA contamination because of the presence of pseudogenes. Depending on the target sequence there may be a few (<10) to a lot (>50) potential pseudogenes that will be amplified with cross intron primers. It may be easier to remove this "contamination" with Taqman based probes than with SYBR green reactions.
Hopes this helps:
Or it can be effectively done by the use of Shrimp Nuclease, which allows you to skip the chemical inactivation. A short Heat inactivation step does the trick.
Since it is dsDNA specific, you can even add the Shrimp nuclease together with the RT, and forget about it, because it inactivates together with the RT.