16S rRNA primers for E. coli - (Feb/07/2006 )

Hi there.

I am a newbie with RT-PCR, and I am trying to use relative quantification to compare my unknown gene from an E. coli to an internal standard under different growth conditions. The internal standard I have been using is glyceraldehyde-3-phosphate DH, but I am getting differential expression between conditions. Someone suggested that I try 16S rRNA instead, but I have no idea which E. coli 16S gene to design primers from or if there is a reference out there with good primers already designed? Otherwise, if I can't get consistent expression for my internal control, what are my options??

Any help is greatly appreciated!!

Tim

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Hi Tim,

E.coli only has one 16S gene, but there are 7 identical copies (one in each rRNA operon). For your purposes this isn't important, as you're interested in using the gene product (16S rRNA) as a reference.

You could try these primers:

357f 5'CTCCTACGGGAGGCAGCAG>
519r 5'GWATTACCGCGGCKGCTG>

These are 'classic' universal 16S primers, from:
Lane, D. J. (1991). 16S/23S rRNA sequencing. Nucleic acid techniques in bacterial systematics. E. Stackebrandt and M. Goodfellow, eds. New York, NY, John Wiley and Sons: p115-175.

There should be plenty of recent references describing use of bacterial 16S rRNA as a reference for gene-expression studies. Alternatively, read up on use of 18S rRNA for eukaryotic assays - the principles are the same.

These primers should work perfectly with E.coli; use the downstream (519r) primer for RT, giving an RT-PCR product of 162bp, which should be fine for qPCR. The convention for numbering of 16S primers corresponds to the position of the 5' end in the E.coli 16S gene, by the way.

good luck.

Del.

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Thanks Del,

This is exactly what I needed to know!

Tim

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