qPCR RT experiment set up

Question

[Maria17]

Hi All,

Currently, I'm working on a qPCR assay. I am using RT kits for my experiment . the setup that i had before for my assay is this :

For 1 Reaction: Add 2 RT Buffer + 4.5 H20 + 0.5 spike-ins +1 RT-Enzyme For 25 Reaction: Add 50 RT Buffer + 112.5 H20 + 12.5 spike-ins + 25 RT-Enzyme usually i make MM 10% more.

8 µl MM + 2 µl RNA to the reaction mini tube . i have 5 reaction tube so in total = 50 µl reaction. After RT i have 50 µl cDNA.

I could not detect any target in my qpcr, so i turned back to the handbook to re-setup my experiment .

in the handbook they mentioned these points:

• Use a template RNA volume equivalent to 16 µl original serum/plasma for each 20 µl reverse transcription reaction.
• Use a template RNA volume equivalent to 8 µl original serum/plasma for each 10 µl reverse transcription reaction. i have 2 quastions:

1:I do not know what is the best experiment set up according to my previous set up.is amount of cDNA enough?or how i optimize this assay?

2: is the RT calculation correct ?

Any recommendations or solutions would be appreciated.

Thanks

Maria

[Trivas]

Are there primers in the RT buffer or is the kit expecting you to use your own primers?

[Maria17]

Dear Travis , yes there are primers in the 384 customized plate.so i did not add them.

Regards Maria