Mircury sybr green pcr kit

Total RNA was extracted from the ischemic brain or cultured cells using TRIzol LS reagent (Invitrogen, Carlsbad, CA) and reversely transcribed into cDNA by ZymoScript II First Strand cDNA Synthesis Kit (Abconal, Shanghai, CN) according to the manufacturer's instructions. qRT-PCR was performed on a 7900HT (ABI, Foster City, CA) with the following condition: 95 °C for 30 seconds followed by 40 cycles of 95 °C for 5 seconds and 60 °C for 30 seconds. GAPDH was used as the internal reference. For EVs' miRNA, the total miRNA was extracted using miRNeasy Serum/Plasma Kit (Qiagen, GER) and transcribed into cDNA using miRCURY LNA RT Kit (Qiagen, GER). The real-time PCR was performed using miRCURY SYBR Green PCR Kit (Qiagen, GER) according to the kits' instructions. The information of mRNA and miRNA sequences are in the supplementary materials.

We used following TaqMan primer and probes (Applied Biosystems, Foster City, CA, USA) SMAD3 (Hs00969210_m1), STAT3 (Hs00374280_m1), STAT5a (Hs00559647_m1), SOCS1 (Hs00705164_m1), GAPDH (Hs02786624_g1), and RPLO (Hs99999902_m1). The following assays were used in microRNA experiment: hsa-miR-24-3p (QG-339306_YP00204260), hsa-miR-26a-5p (QG-339306_YP00206023), hsa-miR-31-5p (QG-339306_YP00204236), hsa-miR-100-5p (QG-339306_YP00205689), hsa-miR-126-3p (QG-339306_YP00204227), hsa-miR-146a-5p (QG-339306_YP00204688), hsa-miR-155-5p (QG-339306_YP00204308), hsa-miR326 (QG-339306_YP00204512), SNORD48 (hsa) (QG-339306_YP00203903), and U6 snRNA (has, mmu) (QG-33906_YP00203907). To prepare quantitative Real-Time PCR, we used TaqMan Gene Expression Master Mix (Applied Biosystems); and for quantitative with microRNA, we used miRCURY SYBR Green PCR Kit (Qiagen). Quantitative Real-Time PCR was performed on real-time cycler (Quant Studio 5, Applied Biosystem, Foster City, CA, USA). Each sample was analyzed in duplicates. From that, we took the mean Ct value, and we used it in the next steps of the analysis. Ct values higher than 35 were taken out of analysis and considered below quantification. The housekeeping gene has been selected and relative expression was calculated by ΔΔCt method or ΔCt method normalized to RPLO; in the case of microRNA, SNORD48 was used as a reference.

Two-step quantitative real-time PCR (qRT-PCR) analysis was performed to assess miRNA expression levels in barley varieties “Marthe” and “KWS Olof” grown with different concentrations of Fe₃O₄ and CuO NPs compared to control plants. miRNAs were extracted from the shoots using a Universal RNA/miRNA Purification Kit (EURx, Poland). RNA was extracted from 140 samples from “Marthe” and “KWS Olof” variety fresh shoots (20 in each experimental group). RNA was quantified and qualified with a spectrophotometer (NanoDrop One, Thermo Scientific, USA). Samples with an A260/280 ratio from 1.7 to 2.1 were used for analysis. miRNA target-specific primer hvu-miR156a with locked nucleic acid was designed. The target miRNA hvu-miR156a sequence was 5′-TGACAGAAGAGAGTGAGCACA-3′. HvsnoR14 was used as a reference gene for the normalization of miRNA expression values. Reverse transcription for miRNAs was performed using the Thermal Cycler UNO96 (VWR, United Kingdom) and miRCURY LNA RT Kit (Qiagen, Germany) according to the protocol first-strand cDNA synthesis. For miRNA qRT-PCR analysis, miRCURY SYBR Green PCR kit (Qiagen, Germany) was used according the manufacturer's protocol. The Rotor Gene Q Series Software program was used to analyse the miR156a expression level of barley varieties. The results were analysed using the 2 − ΔΔCT method [40 (link)].

Total RNA was extracted from cells using TRIzol reagent (15596026; Invitrogen, Carlsbad, CA, USA). The q-PCR was employed to detect the expression levels of DEGs. Total RNA was extracted from each sample. GAPDH or U6 were used as internal reference genes. The 2^−ΔΔCt method was used to calculate the relative expression level of the target gene. The q-PCR was performed using miRCURY SYBR Green PCR Kit (Exiqon–Qiagen) according to the manufacturer's instructions with StepOnePlus Real-Time PCR System (ABI, Thermo Fisher Scientific). Each experiment was repeated at least three times. The primers used for the q-PCR are listed in Table 1.