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.... RNAi: Frequently Asked Questions
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- Why does EZBiolab only provide PAGE purified siRNA oligos?
"Don't waste a clean idea by a dirty enzyme", said one of the greatest biochemists
of the 20th century. This certainly applies to siRNA too.
Since the length and the sequence of a siRNA are critical for the specificity and
efficiency of its gene silence effect, there is no question that siRNA used in every
experiment should be as pure as possible. PAGE is one of the most reliable way
to get high purity RNA oligos. Also, PAGE can catch a single mismatched base pair
from double strand siRNA. So PAGE purification makes sure that a siRNA is not
only pure, but also accurate.
- How effective is your TripleStrike package?
Our TripleStrike package has achieved >98% success rate. Among them,
* all three siRNAs silenced target genes: 18%
* two of three silenced target genes: 56%
* one of three silenced target genes: 28%
In the rare cases (<2%) that all three siRNAs failed to silence a gene, the main
reasons were high GC content or high homology of the target gene sequence to
other genes which resulted non-specific silence.
- How much synthetic siRNA oligos do I need for a gene silence experiment?
In general, 1 OD (~2.5 nmol) of siRNA can supplies 50-70 wells of 24-well cell
culture plate. If tranfection efficiency is not a problem, 10 nM siRNA is sufficient
to silence its target gene.
- Do you have any suggestion about siRNA transfection?
- Can you design siRNA based on protein sequence?
No. DNA sequence is essential for siRNA design
- What is difference between H1 and U6 promotors?
Both are RNA polymerase III promotors. In term of siRNA transcription efficiency,
we find little difference between the two in CHO cell, HEK293 cell and Hela cell.
- Do Human H1 and U6 promotors also work on rat or mouse cells?
Yes. The two peomotors are not limited to human cells, They also work on other
mammalian cells, such as rat and mouse.
- Which tranfection reagent do you recommend for siRNA oligos?
We find those by Qiagen, such as PolyFect and SuperFect are quite effective. Some
others, such as Lipofectamine 2000 by Invitrogen are also very effective.
- For a target site that has already been proven by a synthetic siRNA, does a lentiviral
shRNA give the similar degree of gene silence results?
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