Rneasy mine kit

Shrimp total RNA was extracted using RNeasy Mine Kit (Qiagen, Germany) and reverse transcribed into cDNA using a PrimeScript^TM RT reagent kit (TaKaRa, Japan). A partial cDNA sequence homologous to mammalian IRFs was retrieved from the sequenced L. vanname transcriptome data56 (link), and primers IRF-5RACE1 and IRF-3RACE1 were then designed to receive the 3′ and 5′ ends of IRF cDNA sequences by rapid amplification of cDNA ends (RACE). PCR program was set as described before57 (link). The PCR products were used as templates with the primers IRF-5RACE2 and IRF-3RACE2 for the secondary PCR, with the PCR conditions the same as above. The PCR products were then cloned into the pMD-19T vector (TaKaRa, Japan) and sequenced. The sequences were analyzed and deposited in the NCBI GenBank (GenBank accession no. KM277954).

LruC gene expression was determined in mice organs samples by qPCR after extraction of RNA and synthesis to cDNA [19 (link)]. Total RNA was extracted using RNeasy mine kit (Qiagen) according to the manufacturer’s instructions and quantified in a NanoDrop 1000 spectrophotometer. Quantitative PCR was performed in a total reaction volume of 12.0 μL, containing 0.4 μL of each LruC or 16S primer, 6.25 μL real-time PCR SYBR green Master Mix (Applied Biosystems), 1 μL of cDNA template of 1:10 dilutions and sterilized deionized water up to the final reaction volume. All qPCRs reactions were performed in triplicate using the primers forward-ACGCACAAACCGGCTATAA and reverse-CGGGAATACCTTTGCTTGATTG for LruC gene, and the primeres forward-TTCAGTTGGGCACTCGTAAG and reverse-CGTGTGTTGCCCTAGACATAA for Leptospira gene 16S as endogenous normalized. The qPCR assay was performed and analyzed using Applied Biosystems 7300 Real-Time PCR system and its efficiency was determined for each reaction using software LinRegPcr. All oligonucleotides had the correlation coefficient squared (R2) higher or equal to 0.998 and their efficiency range was in between 1.9–2.0, which indicates stable and reliable assay.