Iscript rt pcr kit

Total RNA was isolated from human umbilical cord mesenchymal stem cells using Trizol reagent (TransGen Biotech, Beijing, China). With iScript RT-PCR Kit (Bio-Rad, California, USA), 1 μg of purified RNA was used for reverse transcription. QPCR analysis was performed using SYBR Green PCR Master Mix (Bio-Rad, California, USA). The expressions of 18S, PGC-1α, Nrf2 and Sirt3 were detected by real-time quantitative PCR. The expression of the target gene was calculated using the 2-^△△Ct method and normalized to 18S.

We used the RNAeasy kit (QIAGEN) and IScript RT-PCR kit (BioRad, Hercules, California, USA) to extract the total RNA and prepare the cDNA, respectively. The qPCR was performed using SYBR^® Premix Ex Taq II (Applied biosystem and Life technology, Waltham, MA, USA) and set up in a 7500 Fast Real-Time PCR system (Applied Biosystems, Foster City, CA, USA). The primers were purchased from Integrated DNA technologies (IDT, Coralville, IA, USA).
SMAD7
Forward primer: 5′-CTTCTCCTCCCAGTATGCCA-3′
Reverse primer: 5′GAACGAATTATCTGGCCCCT-3′
Serpine 7 (PAI-1)
Forward primer:5′- CAGCATGTTCATTGCTGCCC-3′
Reverse primer: 5′GGAGAGGCTCTTGGTCTGAAA-3′
PLEXIND1
Forward primer: 5′-GCTGGCCCATTCAAGATCC-3′
Reverse primer: 5′-GCACCAAATGGAAATACTTCTCTGT-3′

Independent triplicates (n = 3) of 44.000 human brain pericytes were cultured in 4 well plates (24-plate well size) and grown for 20h. The cells were then washed with PBS. Cells were incubated in normoxia, hypoxia or OGD as described above for 2 or 6 hs, respectively. Then 300μl of RLT-plus-lysis buffer (Qiagen) was added. The lysate was frozen in -20°C prior to RNA isolation. Total RNA was isolated using the Qiagen RNA mini plus kit and protocol. A Nanodrop 3000 was used to measure the quality and quantity of the RNA. The RNA was then reverse transcribed with the iScript RT-PCR kit (BioRad) with reactions containing reverse transcriptase (RT) and without RT (-RT). The cDNA was diluted from 20μl to 150μl to be able to load 5μl cDNA per qPCR reaction. QPCR was run in 20μl reaction on a BioRad qPCR-machine with the SSO-advanced qPCR-SYBR mix according to the manufacturers instructions (BioRad). The fold changes between treatments were normalised to the B2M house keeping gene shown to be stable in hypoxic conditions [38 (link),39 (link)]. The primers for the qPCR were designed by primer 3 in the NCBI primer selection software and are available on request. Primers were from TAGC Copenhagen. The qPCR data was analysed in Graphpad Prism and statistic significance was assessed with 2-way ANOVA and Dunnett’s multiple comparison test.

Ticks were allowed to feed for 24 h, for 72 h, or to repletion (between 80 and 96 h after initiation of tick feeding) and RNA isolated from guts and salivary glands using Trizol (Invitrogen, CA) as described earlier [22] (link). cDNA was synthesized using the iScript RT-PCR kit (Bio-Rad, CA) and analyzed by quantitative PCR for the expression of tick actin and B. burgdorferi and also ixofin3D, clone 2, 3 and 4 using gene-specific primers (listed in table S1) and the iQ SYBR Green Supermix (Bio-Rad, Hercules, CA) on a Opticon Engine MJ cycler (Bio-Rad, CA).

Total RNA was extracted from mouse whole brain tissue and CECs with TRIzol Reagent (Invitrogen). Two microgram target RNA in each group was converted into complementary DNA (cDNA) by using iScript™ RT-PCR Kit (Bio-rad, USA). The synthesized cDNA products were used for real time PCR with SYBR Green Supermix (Bio-Rad Laboratories) as the fluorescent DNA intercalating agent. The following primers were used in this study: AIP1, 5′-CCTGCGCGTATCAGTCCTTACCA-3′ (forward); 5′-GGGTTCAGAGCCCTCCTC-3′ (reverse); GAPDH, 5′-GGAGAAGGCTGGGGCTCAT-3′ (forward); 5′-TGATGGCATGGACTGTGGTC-3′ (reverse).