Making shRNA plasmid constructs
9/7/05
1. Download mouse and rat (and human if relevant) sequences from the gene data base at NCBI (http://www.ncbi.nlm.nih.gov, search “gene” then enter the gene name and mus for mouse or rattus for rat). Record the gene name, and the mRNA “RefSeq” number (in blue on the left when you are looking at the exon diagram). Click on the RefSeq ID, and select FASTA. Copy the sequence (control A, control C) and paste into a new DNA file in Vector NTI Explorer or keep the sequence as a text file in Word. From the RefSeq GenBank entry, record the coordinates of the cDNA.
2. Align the species sequences in Vector NTI or using ClustalW (many sites – here is one http://pir.georgetown.edu/pirwww/search/multaln.html) so that you can pick oligos that will knockdown both mouse and rat.
3. Take one of the sequences for analysis on the siRNA site of the Whitehead Institute (http://jura.wi.mit.edu/bioc/siRNAext/home.php). Login is username: west@neuro.duke.edu, password:•••••••••••••. Paste your sequence into the text box. Choose NAN21 (type B) as the sequence to search on, this will include AAN19TT sites which are type A. You do not need to change any other parameters. Click “search”.
4. Your results appear in the next window. Print these to compare to the aligned sequences. You can then BLAST to make sure the oligos will not hit other genes. Make sure to choose the correct species before you blast.
5. There are many factors to consider when choosing your oligos. Please read the attached information about the thermodynamics of siRNAs. Most important is that the stability of the 5’ end of the AS oligo is weaker than the 5’ end of the S oligo as only the less stable end is fed into the RISC complex. On this page, this difference in stability is used to rank the sequences, so most theromodynamically different appear at the top of the list. You also want the 5’ end of the AS strand to be not too stable. The thermodynamic stability of the AS strand is the second number in parentheses. The smaller the absolute value of this number, the less stable. I have had good luck with oligos that have –6 or so here. For vectors that use the U6 promoter you must have a 19bp Sense sequence that starts with G. You will also need to look for conserved sequences across species, and may want to consider targeting specific splice variants. You will need at least two independent oligos for any gene knocked down, so may want to start with three possible sequences. In general, people have been using siRNAs against the coding sequence staring about 100bp from the ATG as it was feared there would be RNA binding proteins blocking 5’ and 3’ UTRs and near the start site. However this may not be true (see Tom Tuschl web site for discussion http://www.rockefeller.edu/labheads/tuschl/sirna.html).
6. Plug the sequence into the template required for cloning into your vector of interest.
pLL3.7 and pLL3.8
HpaI to XhoI
Must choose an siRNA sequence that starts with a G for the U6 promoter
Sense oligo: 5’ T-GN18-TTCAAGAGA-[REVERSE 81NG]-TTTTTTC
AS oligo: 5’ TCGAGAAAAAA-GN18-TCTCTTGAA-[REVERSE 81NG]-A
Oligos are 55/59 nt
pSuper
BglII to HindIII
Uses H1 promoter, so more tolerant of first base in siRNA sequence
Sense oligo: 5’ GATCCCC-N19-TTCAAGAGA-[REVERSE 91N]-TTTTTGGAAA
AS oligo: 5’ AGCTTTTCCAAAAA-N19-TCTCTTGAA-[REVERSE 91N]-GGG
Each oligo 64nt
7. Order from IDT (http://www.idtdna.com)
Username:
Password:
Quote
Need to do 100nmol scale, but standard desalting is fine, no PAGE or HPLC needed
Can get 5’ phosphorylated if you want, but not necessary
8. Enter oligos in the lab oligo database. Please enter all information.
1. Start with 4ug of vector plasmid (pSuper or pLLx3.8)
2. Digest first with one of the enzymes to open the vector
Bgl II for pSuper
Hpa I for pLLx3.8
1uL enzyme should be sufficient in a 10uL volume for 1-2 hours at 37 degrees.
3. Follow instructions to purify on a qiagen gel extraction column without the gel (PCR cleanup protocol). Add 5 volumes buffer PB (50uL, from the miniprep kit), load on column, wash with PE (add EtOH first if needed) and elute in 30uL buffer EB.
4. Save a 3uL aliquot of the elution for a diagnostic gel at the end, then cut the other 27uL with the second enzyme.
HindIII for pSuper
XhoI for pLLx3.8
1uL enzyme in a 40 uL volume for 1-2 hours at 37 degrees should work. Adjust buffer and BSA accordingly.
5. Add 1uL SAP to phosphatase the cut ends of the vector to prevent vector religation. Keep at 37 degrees for 1 hour.
6. Heat kill the SAP by heating at 65 degrees C for 1 hour. Transfer to a PCR tube and do using the “instant incubate” function if no 65 degree water bath is available.
7. Gel purify the prepared vector on a 1% agarose gel in TAE with ethidium bromide. You need four samples for the gel. 1: 1kB Molecular weight standards 2uL of undiluted stock or 12uL of working solution. 2: 1ug of the original uncut vector (this should run differently from the cut samples, if not, the enzymes failed), 3: 3uL of the vector that you saved after the first enzyme cut and purification, 4: the whole sample that was cut with the second enzyme and SAPed (40uL). Make the gel with wide combs and try to fit the sample to be purified in 1 or at most 2 lanes. To each sample you will need to add 6X DNA sample buffer (SB) before loading (that is 2uL of SB added to each 10uL of sample). It is hard to pipette less than 10uL easily into a gel, so for samples 1 and 2 add ddH2O to make the sample 10uL then add 2uL of SB.
8. Run the gel until the MW bands are well resolved in the region that reflects the size of the vector. pSuper is 3176 bp, pLLx3.8 is 8315bp.
9. Take a quick photo of the gel to document the sizes and amounts, then cut out the double cut band.
10. Follow the qiagen gel extraction protocol to elute the band from the gel. Note that since the pLLx3.8 vector is very large, you will need to add 1 volume isopropanol to the tube after the band is dissolved in buffer QC before loading on the column. This will improve your yield. Elute in 30uL buffer EB.
11. Run 3uL of the eluted band on a gel with MW standard to ensure that you achieved good recovery.
1. Anneal the S and AS oligos for a single RNAi construct together.
Spin down oligo tubes
Dissolve in 25uL ddH2O ( for 25nM scale, 55-64 bp in length)
Take 1uL of each oligo (S and AS)
Add 48uL annealing buffer (recipe below)
Incubate 4 minutes at 95 degrees by boiling in a 250mL beaker of water.
Remove beaker from the heat plate, and stir in the cold room to slowly cool to 4 degrees (about 60-90 minutes).
Cooled annealed oligos can be stored at –20 degrees.
Annealing buffer (store at –20):
100mM potassium acetate
30mM HEPES-KOH pH 7.4
2mM magnesium acetate
2. Phosphorylate the annealed oligos
Take 2uL of the annealed oligos
Add 1uL T4 polynucleotide kinase buffer
Add 1uL 1mM ATP (stock is 10mM)
Add 1uL T4 polynucleotide kinase
Add 5uL ddH2O
Incubate 30’ at 37 degrees C
Incubate 10’ at 70 degrees to heat kill kinase
Spin down
1. Ligation:
2uL annealed phosphorylated oligos
2uL 5X ligase buffer (aliquot to limit freeze/thaw)
1uL prepared vector
4uL ddH2O
1uL ligase
incubate 1hour at RT or 4 hours at 16 degrees
2. Transform half of ligation into bacteria following standard protocol. For pLLx3.8 use Sure or Top10 cells as the vector is prone to recombination.
3. Pick 6 colonies of each transformation and grow up for minipreps.
4. Check miniprep DNA for correct ligation.
pSuper:
1) cut BglII run on 1% gel. Choose only colonies that are NOT cut, since BglII site is lost with correct ligation. Presence of BglII indicates original vector. Make sure to run uncut vector DNA and cut vector DNA on the gel when you analyze so you know what that looks like.
2) Cut EcoRI and HindIII. Positive is 360bp and negative is 300bp. You will need at least a 2% gel to resolve these, and run 100bp markers. Make sure to run a positive (pSuper with insert) and negative (pSuper only) control.
pLLx3.8:
cut EcoRI and Xho (will need to look this up…)
5. Send up to three positives for sequencing.
pSuper primers:
pLLx3.8 primers:
6. Grow a maxiprep of any positives you intend to use for transfection or viral production, then cut and sequence again to make sure they are correct.
V. Initial validation:
1. Transfect cDNA for the gene you wish to knock down into 293T cells. Cotransfect either empty vector DNA (pSuper or pLLx3.8) or the vector with your specific shRNA. Use a 20:1 (w/w) ratio of RNAi to cDNA. Wait 2 days, make