Hi,
I am trying to design siRNA for a gene knockdown study, and for this, I am using the tool offered by GenScript (linked below). However, I noticed that the input for the sequence is "cDNA." When I go to the GenBank Database offered by the National Institute for Biotechnology Information (also linked below), I noticed that the mRNA sequence provided for my gene of interest is over 11,000 base pairs long and has thymines rather than uracils. Is this the sequence for the cDNA of the gene? If so, does it contain introns? The Open Reading Frame for this gene is only about 1,000 base pairs in comparison. If this does contain introns, then would it still be fine to use as an input for the below tool? Or should I stick to the Open Reading Frame sequence? My apologies for so many questions, but thank you all for your help!
GenScript siRNA design tool: https://www.genscript.com/tools/sirna-target-finder
GenBank Database for National Institutes of Biotechnology: https://www.ncbi.nlm.nih.gov/nuccore/NM_001145306.2
The sequence you linked to from GenBank is the cDNA for that gene (i.e. the DNA sequence of the entire CDK6 mRNA). Under the "Features" heading of the GenBank entry you will see the coordinates for various annotated regions within the cDNA, including individual exons. Because this is based on the mature mRNA no introns are included in the feature annotations. (If you look at the genomic coordinates for the CDK6 gene you'll see it is 231.6 kb in length while the GenBank entry is only 11.6 kb.
What Lee Yx is suggesting will give you only the cDNA of only the open reading frame. That will work for the siRNA design tool, but you're giving up ~90% of your potential siRNA target sites by using the CCDS rather than the full cDNA.
Hi. cDNA is reverse transcribed RNA. Extract total RNA and by using reverse transcriptase, convert the RNA to cDNA. It will be single stranded DNA derived from RNA. It is used in RTPCR and other PCRs etc
cDNA (Complementary DNA) is a DNA copy of an mRNA (messenger RNA) molecule synthesized by reverse transcriptase in a laboratory/not genomic DNA.
You only need to use the CDS region of your mRNA from the start codon ATG and stop codon TGA. Please copy and paste it into your siRNA design tool, and it means your cDNA that the software requires.
- Badges
- Science topic
- Similar topics
- Biological Science
- Molecular Cell Biology
- Culture Cells
- Cell Line