ABI7000 + SYBR GREEN I
Posted 14 March 2005 - 04:23 PM
I am looking for some good advise... I want to do quantitative PCR in order to determine the expression level of some prokaroytic genes using ABI7000 and SYBR GREEN. When I started reading papers about the whole topic I got a little bit scared since there seem to be so many sources of potential errors (standars, primer dimer, standard curve, dilutions.... .
Does anyone can recommend a good tutorial about the ABI7000 or could provide me with a protocol?
Thanks very much for every response!!!!
Posted 21 March 2005 - 09:27 PM
Since you are trying to determine expression level of certain gene, you have to do a real-time quantitative RT-PCR.
First you have to prepare your standard in RNA form. I would recomemd you to use in vitro-transcription methods rather than cloning.
Go to get an Ambion MAXI script in vitro transcription kit and follow the protocol provided, you will able to get some high abundant and good quality of RNA.
Purify your RNA. Do not use any commercial kit if your fragment is around 100-200bp. Because as the fragment is too small, it woun't stuck on the membrane of the spin column when you spin it. Try to follow conventional method. More useful.
Quantify your RNA using spectrophotometer and convert them into copy number, you should able to get around X e11 copy of RNA.
Aliquot you stndard stock and store in -20. Newly prepare a 10 fold serial
dilution each DAY when you run your assay. The remainder put into +4 and you can use within the same day.
I am using BioRad iCycler, so can not give you any info about ABI prism.
I hope it work for you
Posted 22 March 2005 - 08:44 AM
Thanks very much for your very helpful advise! As for the internal standard preparation,
I run across different papers suggesting either 16S rRNA or in vitro transcribtion.
Did you make any experiences with 16S rRNA as an internal standard? It seems for me more obvious to use RNA as a standard since I am looking at the mRNA level in the cells.
Posted 22 March 2005 - 09:27 PM
Yes, you are right, since you are quantifying gene expression level, the best way of doing is measuring the expression of housekeeping gene, so that you can normalized the signal of both genes.
All the while I have been doing absolute quantification, but not relative quantification (using internal control). Sorry I can't give instraction in detail, i am sorry about that.
However, when you using SYBR Green, is hard to differentiate two PCR product by melting curve analysis, unless both of the GC% of your both genes are distinct enough.
Posted 24 March 2005 - 01:33 PM
I would add this: it is not difficult to determine the difference between two pcr products if you are not using multiplex reactions.
Good references: ABI's user bulletin #2 (hard to find on the website, but more useful than anything else they have published). Also, Qiagen and Ambion have some very good resources online.