There are several steps in generating hairpin primers. First, a 29nt “sense” sequence which ends with a “C” is picked out from the coding sequence of gene of interest. Second, the actual hairpin is constructed in a 5’→3 orientation with respect to the intended transcript.
Predicted shRNA structure
Third, a few stem pairing are changed to G-U by altering the sense strand sequence. G-U base pairing seems to be essential for stability of short hairpins in bacteria and does not interfere with silencing. Finally, the hairpin construct is converted to its “reverse complement” onto which is added 21nt of homology to the Human U6 promoter.
All of the aforementioned steps are automated using a program developed by Ravi Sachidanandam and Jeremiah Faith (CSHL) where either accession numbers from GenBank or raw sequences are required to generate hairpin PCR primers.
[Note: Don’t let the G-U pairings represented in the primer fool you into thinking the primer is incorrect. ]
A link to the hairpin primer generation program, the “RNAi oligo retriever”, can be found at:
http://katahdin.cshl.org:9331/RNAi/html/rnai.html
Make sure that you enter accession numbers and sequences which match cDNA or exon sequences!
THE PROTOCOL
Ordering Primers
Since very little primer is required for the PCR reaction they can be ordered .05μmol scale from Sigma-Genosys or whomever. We find that PAGE purification is costly and unnecessary (PCR will fill in shorted primers!).
PCR
We use a pGEM1 plasmid containing the human U6 locus (N. Hernandez, CSHL) as the template for the PCR reaction. This vector contains ~500bp of upstream U6 promoter sequence. Since an SP6 sequence flanks the upstream portion of the U6 promoter, we use an SP6 oligo as the universal primer in U6-hairpin PCR reactions.
SP6 sequence: GATTTAGGTGACACTATAG
We have had consistently good results using Taq polymerase for PCR with 4% DMSO and 50pmoles of each primers. (For pENTR/D-Topo cloning [see below], I add .1uL of Vent to polish the ends.)
PCR conditions: 95° for 3 min; 30 cycles of 95° for 30 sec, 55° for 30 sec, & 72° for 1 min; followed by one cycle of 72° for 10 min.
