I diluted my cdna 1: 5 times . I added 500ng of rna for cdna conversion so I presume tat I have 500ng of cdna. In 1: 5 dilution my gapdh ct value was 16 to 21 range. But my target gene dint work out. Can I use undiluted cdna
Good, but there is no shortcut answer why do we dilute the cDNA?. I am going to do QPCR. What do you advise me? Shall I dilute the cDNA sample before or not? if yes by how much times? Thanks
I have a problem: my control (GAPDH) amplifies with Ct-s between 24-30, but my target is above 34 and in some samples it does not amplify. Nothing changes if I add more cDNA, even adding up to 100ng/rxn.
my Gaps is very low compared to my other genes in relative real-time per. I started with 1:5 dilution of cdna. my applicants size of target gene mcl1 and gaps is 112 and 510 respectively
Do you mean that the Ct is very low, or the expression is very low? If you are using a sample and assay for the first time, it is helpful to run a dilution series (5 points, 10-fold dilutions) to assess the linear range of your assay and the best working dilution to use. You can reach us at techsupport@thermofisher.com for more assistance.
Your amplicon for gapdh is also quite large. You should choose primers that will give you an amplicon from 90 - 180 bp. Your target gene amplicon looks good.
It’s best to refer to the recommendations of the particular 1-step kit, as they may have different ranges. Many kits can accept anywhere from 1 pg up to 100 ng of undiluted RNA.
I used the same samples to test gene expressions then stored the cDNA in -20degree, the first RT-PCR showed 3 fold increase for two samples. Use the same samples 3 days later, the RT-PCR showed 1.9 ~ 1.5 fold change . what seems to be the problem ? even some genes were increased now they tend to have bit decreased. . Thank you
These are many possibilities for why the expression levels may appear to change. Here are the most common ones you can check for. Are you using the same threshold per gene target across all of your runs? Have you validated that the endogenous control gene is stably expressed across all samples? You can reach us at techsupport@thermofisher.com for more assistance.
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Excellent video, thankyou
great clarity
I diluted my cdna 1: 5 times . I added 500ng of rna for cdna conversion so I presume tat I have 500ng of cdna. In 1: 5 dilution my gapdh ct value was 16 to 21 range. But my target gene dint work out. Can I use undiluted cdna
Hello, it is my first time to use qRT-PCR, any advice before to start?
Good, but there is no shortcut answer why do we dilute the cDNA?. I am going to do QPCR. What do you advise me? Shall I dilute the cDNA sample before or not? if yes by how much times? Thanks
I have a problem: my control (GAPDH) amplifies with Ct-s between 24-30, but my target is above 34 and in some samples it does not amplify. Nothing changes if I add more cDNA, even adding up to 100ng/rxn.
I have the same control but haven't started workflow yet. I'm afraid to face the same issue!!
Awesome, thank you!
my son in law works with you guys at Thermo fisher
my Gaps is very low compared to my other genes in relative real-time per. I started with 1:5 dilution of cdna. my applicants size of target gene mcl1 and gaps is 112 and 510 respectively
Do you mean that the Ct is very low, or the expression is very low? If you are using a sample and assay for the first time, it is helpful to run a dilution series (5 points, 10-fold dilutions) to assess the linear range of your assay and the best working dilution to use. You can reach us at techsupport@thermofisher.com for more assistance.
Your amplicon for gapdh is also quite large. You should choose primers that will give you an amplicon from 90 - 180 bp. Your target gene amplicon looks good.
what happens if I use a one step kit to do the real-time? how much should I dilute the RNA?
It’s best to refer to the recommendations of the particular 1-step kit, as they may have different ranges. Many kits can accept anywhere from 1 pg up to 100 ng of undiluted RNA.
Hi I use the AgPath-ID™ One-Step RT-PCR Kit
I used the same samples to test gene expressions then stored the cDNA in -20degree, the first RT-PCR showed 3 fold increase for two samples. Use the same samples 3 days later, the RT-PCR showed 1.9 ~ 1.5 fold change . what seems to be the problem ? even some genes were increased now they tend to have bit decreased. . Thank you
These are many possibilities for why the expression levels may appear to change. Here are the most common ones you can check for. Are you using the same threshold per gene target across all of your runs? Have you validated that the endogenous control gene is stably expressed across all samples? You can reach us at techsupport@thermofisher.com for more assistance.
Thank you a lot