When we speak about PCR efficiency to be close to 1 does it mean the same as 100%.
I want to perform RT-qPCR with bovine blastocyst to analysis some genes related with metabolism. Does a pool of 10 blastocyst is enough for the analysis? Does H2AFZ is the best housekeeping to use ?
Thank you
Yes a PCR efficiency close to 1 is the same as 100%. Generally speaking you should strive for a reaction efficiency of 90-105%. A low efficiency can indicate poor primer design, bad reaction conditions or pipetting error (including poorly calibrated pipetters) and a high efficiency is often a sign of inhibitors.
It will depend on the genes that you are looking at, but normally 10 blastocytes should be enough. As for H2AFZ, the reason it is used is that its levels of expression are only slightly increased from the egg to blastocyst stages (generally by approximately 10-fold), where as most other housekeeping gene expression levels go up more than 100-fold. I would however recommend trying a few other housekeeping genes such as YWHAZ, GAPD and SDHA, as it is always better to have more than one (MIQE guidelines suggest at least two).
Yes a PCR efficiency close to 1 is the same as 100%. Generally speaking you should strive for a reaction efficiency of 90-105%. A low efficiency can indicate poor primer design, bad reaction conditions or pipetting error (including poorly calibrated pipetters) and a high efficiency is often a sign of inhibitors.
It will depend on the genes that you are looking at, but normally 10 blastocytes should be enough. As for H2AFZ, the reason it is used is that its levels of expression are only slightly increased from the egg to blastocyst stages (generally by approximately 10-fold), where as most other housekeeping gene expression levels go up more than 100-fold. I would however recommend trying a few other housekeeping genes such as YWHAZ, GAPD and SDHA, as it is always better to have more than one (MIQE guidelines suggest at least two).
Yes, it is just a matter of whether you want to multiply your efficiency factor by 100%. Much greater or lesser than the 90-105% (or 0.9-1.05) bounds indicates that there is something going awry with the reaction and can be equipment error, design error, or something else.
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