Sybr green fast mastermix

About 5 mg total RNA was extracted from the microglial cell line or brain tissue in the affected area with Trizol (Qiagen, Hilden, Germany). And then, Superscript III First-Strand Synthesis SuperMix (Invitrogen, Carlsbad, CA, US) was applied to transcript RNA as cDNA. cDNA and SYBR GREEN FAST mastermix (Qiagen) was adopted for RT-qPCR, as follows: at 95°C for 3 min, at 95°C for 30 min, at 55°C for 1 min, for 40 cycles. Each sample was provided with three secondary holes. All primers used in the RT-qPCR were purchased from Sangon Biological Co., Ltd. Data were collected from the RT-qPCR system (Bio-Rad, Hercules, CA, USA), with GAPDH as an internal control. The relative quantitative value of each gene was calculated with the comparative cycle threshold method. All experiments were repeated three times, and the target gene was calculated by 2^−∆∆Ct. See Table 1 for primer sequences.

Total RNA was extracted from brain tissue 24 h after ICH around the hemorrhage using Trizol (Qiagen, Hilden, Germany) according to the manufacturer’s protocol, afterwards, RNA was reverse transcribed into cDNA using Superscript III First-Strand Synthesis SuperMix (Invitrogen, Carlsbad, CA, USA). Quantitative real-time polymerase chain reaction (q-PCR) was performed using synthetic primers listed in Part 3 of the Additional file 1 and SYBR GREEN FAST mastermix (Qiagen). Data collection was performed on the RT-PCR System (Bio-Rad, Hercules, CA, USA). GAPDH was used as an internal control. The relative quantitation value for each gene was performed using the comparative cycle threshold method [41 (link)].

Total RNAs were isolated from H9c2 cells or myocardial tissues using a RNA Extraction Kit (Promega, Madison, WI, USA) and then reverse transcribed into cDNAs using a cDNA Synthesis Kit (APExBio, Houston, TX, USA). Using a SYBR Green FAST Mastermix (Qiagen, Dusseldorf, Germany), qRT-PCR was performed on ABI7500 system (Applied Biosystems, Foster City, CA, USA). The mRNA expression of related genes was calculated according to the 2^−ΔΔct method and normalized to GAPDH (SNHG4 and DUSP1) or U6 (miR-148b-3p).

The cochleae were quickly isolated, and then TRIzol (Invitrogen, Eugene, OR, USA) was used for total RNA extraction. The Super Script III First-Strand Synthesis System (Invitrogen) was used to synthesize cDNA. Quantitative PCR was performed with the reagent of SYBR Green FAST Mastermix (QIAGEN, Germantown, MD, USA) on an ABI 7300 Sequence Detection System (Foster City, CA, USA). Primer sequences are listed in Table. S1. The cycle reaction was performed as follows: 95 °C for 10 min, 40 cycles of 95 °C for 15 s and 60 °C for 1 min, and finally a dissociation curve of 95 °C for 15 s and 60 °C for 15 s. The gene expression was calculated relative to the housekeeping gene GAPDH and then analyzed using the 2^−ΔΔCT method. Semi-quantitative PCR was performed using the DNA Engine (Bio-Rad, Hercules, CA, USA) with the cycle reaction described above.

Total RNA was extracted from microglia or brain tissues using Trizol (Qiagen, Hilden, Germany) according to the manufacturer’s protocol, after which RNA was reverse transcribed into cDNA using Superscript III First-Strand Synthesis SuperMix (Invitrogen, Carlsbad, CA, USA). The resulting cDNA was used for PCR using SYBR GREEN FAST mastermix (Qiagen) in triplicate. The expression of CD16 and CD206 were detected by RT-PCR using the primers: CD16, Forward: 5′-TCAAATCACTTTCTGCCTGCT-3′, Reverse: 5′-CTATTGCTCTCCTCATCCCAT-3′; CD206, Forward: 5′-AGTGATGGTTCTCCTGTTTCC-3′, Reverse: 5′-GGTGTAGGCTCGGGTAGTAGT3′. All other primers for RT-PCR were used as previously described [14 (link), 20 (link)–23 (link)]. Data collection was performed on the RT-PCR System (Bio-Rad, Hercules, CA, USA). GAPDH was used as an internal control. The relative quantitation value for each gene was performed using the comparative cycle threshold method [24 (link)].