Superscript first strand synthesis system for rt pcr kit

Total RNA was extracted from tissues and cultured cells by using the Trizol reagent (Invitrogen) following the manufacturer's instructions. Purified RNA was reverse transcribed to the first strand of cDNA primed with random hexamers using SuperScript First-Strand Synthesis System for RT-PCR kit (Invitrogen). PCR was then performed at annealing temperature of 58°C for 30 cycles with cDNA as template. The primers were as follows: forward primer 5′-GGAGCACAATGATCCAATCC-3′ and reverse primer 5′-ATGGTTCGA GCATCCATTTC-3′ for IQGAP1 gene. Forward primer 5′-GGCCTCCAA GGAGTAAGACC-3′ and reverse primer 5′-AGGGGTCTACATGGCAACTG-3′ for GAPDH (glyceraldehyde-3 -phosphate dehydrogenase) gene.

Total RNA was prepared using TRIzol reagent (Invitrogen, Carlsbad, CA, USA), according to the manufacturer’s instructions. RNA was reverse-transcribed into complementary DNA (cDNA) by using a SuperScript First-Strand Synthesis System for RT-PCR Kit (Invitrogen), and the cDNA fragments were amplified by PCR with Taq DNA polymerase (Invitrogen). PCR was carried out in a thermal cycler (Bio-Rad, Hercules, CA, USA). Initial denaturation was carried out at 95 °C for 15 min, followed by 40 cycles of denaturation for 10 s at 95 °C, annealing for 30 s at the appropriate temperature, and extension for 32 s at 72 °C. The primer sequences used for PCR were as follows: β-actin, forward 5′-CCTCATGCCATCCTGCGTCTG-3′ and reverse 5′-TTGCTCGAAGTCTAGGGCAACATAG-3′ (annealing temperature: 58 °C); TLR4, forward 5′-GCCTTGAATCCAGATGAAAC-3′ and reverse 5′-CTGTGAGGTCGTTGAGGTTAG-3′ (annealing temperature: 58 °C); NF-κB, forward 5′-TCCCCTGTACGATAGTCGGCTC-3′ and reverse 5′-GAGCGTTGCTTTGGATCAAGG-3′ (annealing temperature: 60 °C); TNF-α, forward 5′-GAAAAGCAAGCAACCAGCCA-3′ and reverse 5′-CGGATCATGCTTTCCGTGCTC-3′ (annealing temperature: 57 °C); and IL-6, forward 5′-CTGGCAATATGAATGTTGAAAC-3′ and reverse 5′-AGAAACCATCTGGCTAGGTAAG-3′ (annealing temperature: 58 °C).

Total RNA was extracted with Trizol reagent according to the manufacturer's guidelines (Invitrogen), and first-strand cDNAs were synthesized by reverse transcription (Superscript First-strand Synthesis System for RT-PCR kit, Invitrogen) Quantitative mRNA expression was measured by real-time PCR with the PRISM 7900 sequence detection system (Applied Biosystems), and the TaqMan master mix kit; EF1-α mRNA was used as an internal control. Human TaqMan gene expression assay for FOXP3 (Hs01085834-m1), CTLA-4 (Hs03044418-m1), GARP (Hs0019436-m1) and EF1-α (Hs01024875-m1) were purchased from Applied Biosystems. The program used for amplification was as follows: 10 min at 95°C followed by 40 cycles of 15 s at 95°C, 1 min at 60°C.

The following were from commercial sources: Trizol™ reagent, Superscript™ First-Strand Synthesis System for RT-PCR Kit, NuPAGE 4–12% Bis-Tris gel, and NuPAGE MES SDS running buffer, N-6-[(7-nitrobenzo-2-oxa-1, 3-diazol-4-yl) amino] hexanoyl-4-d-erythro-sphingosine (C6-NBD-ceramide), Alexa-Fluor555 (Invitrogen, Carlsbad, CA); anti-β-actin monoclonal antibody (Sigma, St. Louis, MO); rat anti-mouse CD68 monoclonal antibody (Serotec, Oxford, UK); M-PER Mammalian Membrane Protein Extraction Reagent and BCA Protein Assay Reagent (Pierce, Rockford, IL); [32P] dCTP (DuPont-New England Nuclear Research Products, Boston, MA); Molecular Dynamics Storm 860 scanner, Hybond™-ECL™ nitrocellulose membrane, and ECL detection reagent (Amersham Biosciences, Piscataway, NJ); antifade/4,6-diamidino-2-phenylindole (DAPI) (Vector Laboratory, Burlingame, CA); FITC-conjugated goat anti-rabbit antibody and rhodamine-conjugated goat anti-rat antibodies (ICN Biomedicals, Aurora, OH); pBROAD3-mcs vector carrying ubiquitous murine ROSA26 promoter (InvivoGen, San Diego, CA); Genomic BAC probe RP23 (BACPAC Resources Center, Oakland CA); Nick Translation Kit (Abbott Molecular, Des Plaines, IL); Hybond N+ nylon membranes (Amersham Bioscience, Freiburg, Germany).

The isolation of total RNA from these seeds using an RNA extraction kit from Invitrogen using the TRIzol method. Assessments of RNA quality and quantity were carried out via 1% agarose gel electrophoresis, complemented by measurements using a NanoDrop Photometer spectrophotometer (IM-PLEN, Westlake Village, CA, USA). This process preceded the reverse transcription of the isolated RNA into cDNA, utilizing the Super-Script™ First-Strand Synthesis System for RT-PCR kit (18091050; Invitrogen, Waltham, MA, USA). Subsequently, the cDNA underwent qRT-PCR (quantitative real-time PCR), with primer sequences meticulously designed as delineated in Table 1. Actin served as an internal control. Primer designs for qRT-PCR were facilitated using Primer Premier 5 software (Biosoft International, Palo Alto, CA, USA). The qRT-PCR experiments utilized the ABI Prism 7900 HT system (Applied Biosystems, Waltham, MA, USA). The ascertainment of relative gene expression levels was achieved via the 2^−ΔΔCT method, with analysis conducted using the 7500 software v2.0.6 (Applied Biosystems).

Retinas were dissected from homozygous r15 mutant mice and total RNA was isolated using the TRIzol® Reagent (Invitrogen Life Technologies). The Superscript™ First-Strand Synthesis System for RT-PCR kit (Invitrogen Life Technologies) was used to synthesize cDNAs. The coding region of the NHE8 gene was amplified by various primer pairs with Platinum® pfx DNA polymerase (Invitrogen Life Technologies). PCR fragments with overlapping regions were sequenced at the UC Berkeley DNA sequencing facility.