I have been working with rabbit bone marrow and periosteal MSCs and inducing the cells into the osteogenic lineage using specific differentiation media.
When performing PCR (so to eventually do qPCR) all of the primers that I used for ALP haven't worked. None! Not the ones I designed and not the ones I found in papers (links listed, but I tried many others!).
There is a mRNA sequence for ALP for rabbit on PubMed (link below) but I don't think it's correct because my colleagues and I designed maybe 5 or 6 pairs in total and nothing works....
If someone has a pair of primers (for qPCR, so preferably not above 200 bp) for rabbit ALP (bone) please let me know or reference me a paper with one that actually works!
Thank you.
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4353779/
http://www.sciencedirect.com/science/article/pii/S1008127515304442?via%3Dihub
https://www.ncbi.nlm.nih.gov/nuccore/XM_017346489.1
Hello Luisa, Will you please send me your primer sequences and conditions you used for amplification.
1 (F) CGTGTTCACCTTTGGAGGAT
1 (R) CTGGGCCTGGTAGTTGTTGT
1 Length of product (bp) – 190
2 (F) GACAAGAAGCCCTTCACAGC
2 (R) ACTGGGCCTGGTAGTTGTTG
2 Length of product (bp) – 118
3 (F) TGTGCGGGGTCAAGGCTAAC
3 (R) GGCGTCCGAGTACCAGTTGC
3 Length of product (bp) – 217
4 (F) AGGCAGGATTGACCACGG
4 (R) TGTAGTTCTGCTCATGGA
5 (F) GGTAAGCGCAGCCACTGAG
5 (R) TCACGTTGTTCCTGTTCAGC
6 (F) GGACATGCAGTACGAGCTGA
6 (R) TTGTCCGTGTCACTCACCAT
7 (F) GACAAGAAGCCCTTCACAGC
7 (R) ACTGGGCCTGGTAGTTGTTG
7 Length of product (bp) – 180
8 (F) CGTGTTCACCTTTGGAGGAT
8 (R) CTGGGCCTGGTAGTTGTTGT
9 (F) CGTGTTCACCTTTGGAGGAT
9 (R) ACTGGGCCTGGTAGTTGTTG
10 (F) TCGCTTATCAGCTCATGCAC
10 (R) CCAGACAAAGTGGGAGTGCT
11 (F) ATCCGGAACATTGACGTGAT
11 (R) AGTGCGGTTCCAGACAAAGT
12 (F) TGCAAGGACATCGCTTATCA
12 (R) GCTTGAAGCTCCTCCAGATG
13 (F) CGCAGGCAATGCTTTTCG
13 (R) CGGGGTCACTGGTATCGTTCT
13 Length of product (bp) – 293
Conditions 95℃ for 2 min, 40 cycles 95℃ for 30 s, Primer Temp for 20 s, 72℃ for 30 s, 72℃ for 2 min (then 4℃ ∞). Since each primer has its own ideal annealing temperature, when testing multiple primers at once then the lowest temp (minus 2 degrees) would be used. That would vary between 61℃ and 63℃.
Some primers I designed, some I got from papers and others the research assistant in the lab designed.
Thank you!
I will suggest you to design primer 100-110 length maximum and try to keep AT sequences at 5 prime of the primer and CC or CG at 3 end of primers.
https://eu.idtdna.com/PrimerQuest/Home/Index
You can above tool to find out primers. I prefer to design manually.
Dear Luisa.
Primers from both the papers you mentioned are not binding to the bone specific ALPL predicted sequence. Blast results shows binding to intestinal ALPI.
Like you, i designed two primers pairs using the bone/kidney alpl rabbit predicted sequence. But nothing worked
If you found the correct primers for rabbit alpl. Will you please share with me. I m doing similar experiment using bone marrow MSCs.
Thanks
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