mRNA expression study on small amount of Theileria annulata lymph node biopsy sample using SMART-cDNA technology

ABSTRACT

A major issue in many gene expression studies utilizing small amount of biological materials is the limited quantity of RNApurified from clinical samples, which is often used for RT-PCR or standard Northern blot analysis. The SMART cDNA synthesis method and subsequent SMART-cDNA-PCR technique was used to analyse 3 genes in macroschizonts of Theileria annulata in small lymph node biopsy material. The SMART-cDNA of TaSp gene was cloned in pTZ57R/T-vector and sequenced. We focused on genes encoding surface proteins TaSp, TaD and HSP70. Our results showed that SMART cDNA dependably reproduces the expression profile found in messenger RNA. The RT-SMART-PCR showed the amplification of the processed mRNAs. The sequencing analysis showed that the amplified cDNA was coded for TaSp protein in Theileria annulata. It was concluded that the SMART PCR technique is practical for amplification of complete sequence of mRNAs in the form of cDNAs, and therefore for gene expression studies if only small amounts of starting material are available