Dntp mixture

Transcripts encoding VEGFR-1, -2, -3, CD31, CXCR4, SDF-1, and β-actin were quantitated by real-time (RT)-PCR analysis. Total RNA from ischemic tissues was extracted with TRIzol reagent (Invitrogen, Carlsbad, CA, USA), and the amount of RNA was measured with a BioPhotometer (Eppendorf Co. Ltd., Tokyo, Japan). Synthesis of first-strand cDNA from total RNA was performed with 1μg of total RNA, 200 U of reverse transcriptase (ReverTra Ace, Toyobo, Osaka, Japan), 40 nmol of dNTP mixture (Toyobo), 20 U of RNase inhibitor (Toyobo), and 20 pmol of oligo(dT)₂₀ in a total volume of 40μL. The reactions were incubated initially at 30°C for 10 minutes, and then at 42°C for 40 minutes, followed by inactivation at 99°C for 5 minutes. Real-time PCR primers were designed using the Primer3 software (http://primer3.sourceforge.net/) using data from GenBank. The DNA sequences of mouse primers used for real-time PCR are described in Table 1.

Total RNA was extracted from the CC using RNAiso plus reagent (Takara Bio Inc.), according to the manufacturer's recommended protocol. The RNA concentration and quality were measured via spectrophotometry at 260 and 280 nm using a Nanodrop 2000 (Thermo Fisher Scientific Inc.). Then, 1 μg of total RNA was reverse transcribed using ReverTra Ace (Toyobo Co. Ltd), dNTP mixture (Toyobo), RNase inhibitor (Toyobo), and oligo (dT)₂₀ (Toyobo) in a TaKaRa PCR Thermal Cycler PERSONAL (Takara Bio). cDNA samples were diluted five times with RNase‐free water. Real‐time PCR was performed with KAPA SYBR green (KAPA Biosystems Inc.) according to the manufacturer's recommended protocol. All primer sequences are shown in Table 1. Initial denaturation was performed at 95℃ for 3 min using a CFX Connect Real‐Time PCR Detection System (Bio‐Rad Laboratories Inc.). Then, amplification was performed for 40 cycles of 95℃ for 3 s and 60℃ for 30 s. The specificity of each primer was confirmed by melt curve analysis. The data were analyzed using the ΔΔCt method, which indicates relative changes in gene expression normalized to the housekeeping gene, β‐actin.

Total RNA of EVs or cells was extracted using TRIzol Reagent (Life Technologies Co., Carlsbad, California, USA). Residual DNA was removed by DNase I digestion following RNA isolation, as described below. The mixture of 11 µl purified RNA and 1 µl of 25 pmol/µl randomized primer (Toyobo Co., Osaka, Japan) in an RNase-free microcentrifuge tube was denatured by boiling at 65°C for 5 min, followed by immediate cooling on ice. Twelve µl of denatured RNA, 4 µl of 5× RT buffer (Toyobo), 2 µl dNTP mixture (10 mM each of dNTPs, Toyobo), 1 µl of 10 U/µl RNase inhibitor (Toyobo), and 1 µl ReverTre Ace (a reverse transcriptase) (Toyobo) were mixed into a total volume of 20 µl transcriptase reagents. The mixture was incubated at 30°C for 10 min, 42°C for 30 min, 85°C for 5 min, and 4°C for 5 min to allow for the synthesis of first-strand complimentary DNA (cDNA). Subsequently, qualitative PCR of cDNA was performed using 2×Taq PCR MasterMix (Tiangen) by the MyCycler thermal cycler (Bio-Rad), as described above.