I experience big discrepancies when calculating ***melting temperature*** of **RNA 7-mers** with Biopython over values generated by a popular algorithm. I tried the nearest neighbour algorithm with RNA and salt concentrations as described in a respective paper (thermodynamic table used as in paper below from: *Freier et al 1986*). Yet, the values largely differ (execute code below to see). I tried all seven salt correction methods provided by Biopython, still I never get close to the values generated by siRNA design algorithm for the same 7-mers. Can someone tell me how accurate Biopython's melting temperature nearest neighbour algorithm is? Especially for short oligomers like my 7-mers? Is there maybe something I am implementing wrong? Any suggestions? Values derived from executing **sample input**: http://sidirect2.rnai.jp/ Tm is given for the seed duplex of the *guide strand*: **bases 2-7** **Literature:** "*Thermodynamic stability and Watson–Crick base pairing in the seed duplex are major determinants of the efficiency of the siRNA-based off-target effect*" http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2602766/pdf/gkn902.pdf from Bio.Seq import Seq from Bio.SeqUtils import MeltingTemp test_list = [ ('GGAUUUG', 21.5), ('CUCAUUG', 18.1), ('CAUAUUC', 8.7), ('UUUGAGU', 19.2), ('UUUUGAG', 12.2), ('GUUUCAA', 14.9), ('AGUUUCG', 19.7), ('GAAGUUU', 13.3) ] for t in test_list: myseq = Seq(t[0]) tm = MeltingTemp.Tm_NN(myseq, dnac1=100, Na=100, nn_table=MeltingTemp.RNA_NN1, saltcorr=7) # NN1 = Freier et al (1986) tm = round(tm, 1) # round to one decimal print 'BioPython Tm: ' + str(tm) + ' siDirect Tm: ' + str(t[1]) Question first asked at stack-exchange: http://stackoverflow.com/questions/30037939/biopython-big-discrepancy-calculating-rna-melting-temperature-over-literature