Northern vs qRT-PCR for expression quantitation - (Sep/14/2005 )
Both Northern and qRT-PCR are often used for quantitative detection of gene expression. I know northern is a golden method. However, the method of qRT-PCR is developing. Do you think Northern still has some advantage than qRT-PCR in term of precision? Thanks.
I tend to believe in Norther more than in qRT-PCR because Northern gives you visual evidence - Seeing is believing.
I agree with pcrman.
Under circumstances favorable (that is: at least 5 microgram total RNA available, no partial degradation of sample), you should choose northern, since signals are directly related to the amount of starting material.
Northerns can tell you transcript size, RT-PCR can not
I prefer qRT-PCR over Northerns.
More things can go wrong with Northerns that prevent easy quantification of transcript (high background, degradation, etc.) and there isn't really a fool-proof control.
And yes, you can tell the transcript size from RT-PCR. Run the cDNA product out on a gel. It's easy and you don't have to worry about Rnase. I do it every day in our lab.
quantitatively, I believe qRT-PCR is more accurate than northen blots
qualitatively, a northen is better a describing the transcripts
Northerns cannot be used reliably to quantitate transcripts. Did you know that you cannot discern a 50% change in quantity on a northern blot by simply visualizing a gel? So, trying to compare your target transcript with a loading control when you can be off by 50% on both means that quantitation is impossible. You could use software that quantitates the bands on your northern, however, once a pixel is saturated, that method becomes invalid. Northerns are very useful in determining the number of transcripts produced and their relative size, even if both are unknown. real-time PCR cannot do that... and to detect multiple transcripts by qRT-PCR you have to design primer sets that will recognize each transcript.
qRT-PCR is much better at quantitating transcripts. The key is to design your experiment appropriately and use a proper reference gene as your "loading control." qRT-PCR is much better than northerns for numerous other reasons, including:
1. you need MUCH LESS sample - I regularly run real-time RT-PCR on 25ng of sample or less, compared to 10ug per lane on a northern
2. No radioactivity - yay!
3. Much faster
4. Less experimental variables to consider - with northerns you worry about transfer, probe labeling, hybridization efficiency, etc...
5. You can compare results experiment to experiment (assuming you use a standard curve in each) - with northerns, you cannot compare one blot to another or one exposure to another.
6. And did I mention, no radioactivity? Yay!