ABSTRACT
Objective To evaluate the stability of U6 and let-7a as internal reference genes of miRNAs in RTqPCR by using femoral head samples of cartilage tissue from inbred DA rats.Methods Total RNA was extracted from femoral head cartilage tissues of female DA rats at three different time points,i.e.at birth (D0),ablactation (D21) and maturation (D42).The expressions of different miRNAs (miR-1,-25,-26a,-140,-146a,-150,-181a,-195,-223 and-337) were detected by RT-qPCR using U6 or let-7a as the internal reference.The two sets of miR expression were compared with the results from Solexa sequencing in our pioneer work to evaluate the stability of the two internal references.Results The relative values of U6 (P =0.045) and let-7a (P =0.021 5) revealed significant difference in the D42 sample.Both in U6 and let-7a systems,miR-26a,-140,-223,and-337 showed a similar tendency in expression and quantification but miR-1 and-146a did not have significant differences.miR-25,-150,-181a and-195 differed significantly (P＜0.05).Comparison of absolute quantification results between the two generations' sequencing showed that let-7a is more stable than U6.Conclusion Let-7a is more suitable to be used as the internal reference gene in RT-qPCR for miRNAs in cartilage tissue.
ABSTRACT
Objective To study the profiles and function of small RNA (sRNA)gene during chondrogenesis in rats so as to clarify the mechanisms of chondrocytes proliferation and differentiation.Methods All the sRNAs were identified from the female SD rats femoral head cartilages at three time points:at birth,ablactation and maturation,and three sRNA libraries were constructed.The Solexa sequencing and the bioinformatics analysis were employed to be blasted with the genomes of SD rats.Results The perfect match reads in the three libraries were screened out,which were correspondent to the 21 7 921 (41.23%),1 96 650 (38.74%)and 245 436 (41.54%)unique sRNA sequence,respectively.The percentages of 20-24 nt sRNA were 71.94% (d0),72.85% (d21),and 86.39%(d42).Half of clean sequences were 22 nt sRNA.The distribution characteristics of the reads were in line with the high-quality sRNA.More than 62% clean reads were from mature miRNA while the ratios in the three libraries were only 0.69%,0.78% and 0.63%.About 60% of the unique sRNA could not be matched with miRBase20.0 or Rfam9.1.Conclusion The distribution model of miRNA in the three libraries indicates that the miRNAs with different functions or from different sources are involved in the regulation of chondrocytes proliferation and differentiation in bone development and formation.