oh hai ya'll i'm new here
So I have a technical problem and hoping that maybe someone here may be able to provide their intellect on the issue.
My lab (we're new/small and the group hasn't started doing benchwork until I joined- previously it's been feeding studies and analysis of old data from my PI's postdoc). Essentially, since i'm the only grad student, I do all of the work- weee. I got trained on PCR by our lab tech, who received her training not too far before me in a cell lab. I recently got trained by our ABI product specialist, and after having some discussions with him i've become worried with the amount of replicates we are running.
So from what i've been able to understand in the past 24 hours, technical samples can get away with running duplicates. However when you're running biological samples you need to run at the very least 3 replicates (because you're looking for differences under 1 cycle). This is already an issue for us because thus far we've only ran duplicates (remember we were trained off cell protocols). And before I start running the PCR for my PI's experiment I will switch to triplicates (in the liver at least).
99% of our brain-work is with the medial basal hypothalamus, I know the brain is a tricky SOB to obtain RNA/DNA/anything from. Because of this, I probably should be using more than triplicates, right? For anyone who does PCR in this area, how many replicates do ya'll do for like membrane receptor expression?
And more specifically, I study cytokine expression in the MBH. I did duplicates, believe me i'm kicking myself now b/c I had 1 gene in particular that was so close to significance, that had I ran more replicates may have become significant. But, cytokine expression varies from cell to cell (as in one may not express anything where others will be expressing like it is its job). So because of this I will not need to use as many replicates, but I was wondering specifically if anyone else in this community has/does look at cytokine expression and how many replicates they use (?).
thanks if anyone can help!!!
My lab (we're new/small and the group hasn't started doing benchwork until I joined- previously it's been feeding studies and analysis of old data from my PI's postdoc). Essentially, since i'm the only grad student, I do all of the work- weee. I got trained on PCR by our lab tech, who received her training not too far before me in a cell lab. I recently got trained by our ABI product specialist, and after having some discussions with him i've become worried with the amount of replicates we are running.
So from what i've been able to understand in the past 24 hours, technical samples can get away with running duplicates. However when you're running biological samples you need to run at the very least 3 replicates (because you're looking for differences under 1 cycle). This is already an issue for us because thus far we've only ran duplicates (remember we were trained off cell protocols). And before I start running the PCR for my PI's experiment I will switch to triplicates (in the liver at least).
99% of our brain-work is with the medial basal hypothalamus, I know the brain is a tricky SOB to obtain RNA/DNA/anything from. Because of this, I probably should be using more than triplicates, right? For anyone who does PCR in this area, how many replicates do ya'll do for like membrane receptor expression?
And more specifically, I study cytokine expression in the MBH. I did duplicates, believe me i'm kicking myself now b/c I had 1 gene in particular that was so close to significance, that had I ran more replicates may have become significant. But, cytokine expression varies from cell to cell (as in one may not express anything where others will be expressing like it is its job). So because of this I will not need to use as many replicates, but I was wondering specifically if anyone else in this community has/does look at cytokine expression and how many replicates they use (?).
thanks if anyone can help!!!