Onestep reverse transcription polymerase chain reaction rt pcr kit

TCR libraries were constructed and next-generation sequencing (NSG) was performed by Chengdu ExAb Biotechnology Co, Ltd as previous report (19 (link)). Briefly, the genomic DNA was extracted using the QIAGEN OneStep reverse transcription polymerase chain reaction (RT-PCR) Kit (QIAGEN), following the manufacturer’s instructions. The PCR products were purified by using Agencourt AMPure XP beads (Beckman). Extracted genomic DNA was accurately quantified using a Qubit 1X dsDNA HS Assay Kit (Invitrogen). The TCRβ complementarity-determining region 3 (CDR3) regions were amplified by Multiplex PCR and sequenced on an Ion GeneStudio S5 System (Thermo Fisher Scientific). The TCRβ CDR3 regions were discerned based on the definition established by the International ImMunoGeneTics (IMGT) Collaboration, and the V, D, J segments contributing to each CDR3 region were identified by a standard algorithm. For each sample, we used the Shannon-Weaver index (20 (link)) and the diversity 50 (D₅₀) value to estimate the TCR diversity. The Shannon-Weaver index reflected the diversity of the TCRs. The D₅₀ index was used as a measure of the diversity and clonality of the TCRβ repertoire and defined as the proportion of TCRβ CDR3 clonetypes that account for the cumulative 50% of the total TCRβ sequences in the sample (21 (link)).

Tissue specimens came from lung tumors, metastatic sites and malignant effusion cell blocks. The process of tissue specimen preparation for EGFR mutation analysis was as described previously [37 (link), 38 (link)]. RNA was extracted from different tissue specimens for gene mutation analysis with Qiamp RNA Mini Kit (Qiagen) according to the manufacturer's protocol. Spectrophotometry was used to quantify extracted RNA.
The cDNA was obtained from the extracted RNA by the Qiagen OneStep reverse transcription polymerase chain reaction (RT-PCR) kit (Qiagen). Exons 18–21 of EGFR were amplified. The tyrosine kinase domain of the EGFR coding sequence, exons 18, 19, 20 and 21, were amplified with forward primer (5′- GGA- TCG- GCC- TCT- TCA- TGC-3′) and reverse primer (5′-TAA-AAT-TGA-TTC-CAA-TGC-CAT-CC-3′) by independent polymerase chain reaction (PCR) amplifications. The associated primers and conditions of RT-PCR have been revealed in our prior published reports [32 (link), 39 (link)]. Finally, EGFR sequences of PCR amplicons were analyzed using ABI PRISM 3100 or 3700 (Applied Biosystems) in both sense and antisense directions.