Lightcycler 480 sw software

PBMCs or CD4⁺ or CD14⁺ cells (2 × 10⁵) were isolated using a FACS flow cytometer (BD Biosciences). The total RNA was extracted from the cells using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. The complementary DNA was synthesized using SuperScript III reverse transcriptase (Invitrogen) according to the manufacturer’s protocol. A LightCycler (Roche, Basel, Switzerland) and a SYBR reverse transcription-polymerase chain reaction (RT-PCR) kit (Toyobo, Osaka, Japan) were used for the quantitative real-time RT-PCR analysis in accordance with the manufacturer’s instructions. The sequences of the primer sets for Sema7A, β1-integrin, and plexin C1, as well as the target sites on the messenger RNA (mRNA) and the PCR product sizes, are shown in Table 1. β-actin was used to normalize the samples. The relative quantification of each gene was determined using LightCycler 480SW software (Roche).Table 1

Primers for human semaphorin 7A, β1-integrin, and plexin C1

Transcript	Forward primer	Reverse primer	Length
Semaphorin7A	TCATCAAAGCCACCATCG	AGCTCACATACAGCTTCCTCC	771
β1-integrin	CAAAGGAACAGCAGAGAAGC	GTGGAAAACACCAGCAGC	537
Plexin C1	AACCATTGCACTGCAAACC	GATTCCATCTTCAAGAATCACG	557

Total RNA was extracted from cells using TRIzol reagent (Invitrogen, USA) according to the manufacturer’s instructions. The cDNA was synthesized using Superscript III reverse transcriptase (Invitrogen) according to the manufacturer’s protocol. A Light Cycler (Roche, Switzerland) and a SYBR RT-PCR kit (Toyobo, Japan) were used for quantitative real-time RT-PCR analysis in accordance with the manufacturer’s instructions. The sequences of the primer sets for the CHIP, alkaline phosphatase (ALP), Ocn and β-actin mRNAs, the target sites on the mRNAs and the PCR product sizes are shown in Table1. The relative quantification of each gene was determined using the LightCycler 480 SW software (Roche).