Kapa sybr fast qpcr kit master mix abi prism

mRNA expression analysis was performed following a previously described method^{5 (link)} with modification. iPS-Cart cells were lysed with Qiazol (QIAGEN), and total RNA was extracted with the miRNeasy Kit (QIAGEN). For real-time quantitative RT-PCR, 50 ng of total RNA was reverse transcribed into first-strand cDNA by using ReverTra Ace (Toyobo, Tokyo, Japan) and an oligo(dT)20 primer. The PCR amplification was performed using the KAPA SYBR FAST qPCR Kit Master Mix ABI Prism (KAPA Biosystems, MA, USA). The PCR primers used are listed in Table 2. The RNA expression levels were normalized to the level of GAPDH expression. Results indicate the relative expression of the molecules (control cells = 1).Table 2

Primers used for real-time PCR.

Primer	Sequence
Human
GAPDH forward	AAGCCCATCACCATCTTCCAGGAG
GAPDH reverse	ATGAGCCCTTCCACAATGCCAAAG
P16(INK4A) forward	GTGGACCTGGCTGAGGAG
P16(INK4A) reverse	CTTTCAATCGGGGATGTCTG
P21 forward	TCACTGTCTTGTACCCTTGTGC
P21 reverse	GGCGTTTGGAGTGGTAGAAA

cDNA of total RNA or immunoprecipitated oxidized RNA was prepared using QuantiTect Reverse transcription kit (Qiagen, Germany). The following PCR program was used for amplification: 15 min at 42 °C, 1 min at 95 °C then at 4 °C. For PCR, Applied Biosystems StepOne™ Real-Time PCR Systems was used. Based on SYBR green reagents (KAPA SYBR FAST qPCR Kit master mix ABI Prism (KAPA Biosystems, South Africa) and the following primer sequences were used:
Aldh2 (F: TGCTACGATGTGTTTGGGGC, R: TTCACTTCTGTGTACGCCTGC), Nqo1 (F: CATTGCAGTGGTTTGGGGTG, R: TCTGGAAAGGACCGTTGTCG), Hgf (F: TGATCCCCCATGAACACAGC, R: CCCCTCGAGGATTTCGACAG), Prdm16 (F: GCCCCATGATGGACAAGACA, R: TCCCAGGATGAGGTCTGGAG), Sod2 (F: TCCGTCCGTCGGCTTCTCGT, R: TCACCGCTTGCCTTCTGCTCG), Pparα (F: TGAGGAAGCCGTTCTGTGAC; R: GTTTAGAAGGCCAGGCCGAT), Cat (F: GCCAATGGCAATTACCCGTC, R: GAGGCCAAACCTTGGTCAGA), 18srRNA (F: TACCGCCCCTCGTAGACAC, R: GCTCTGACCTCGCCACC), actin ß (Actb) (F: CCTTCCAGCAGATGTGGATCA, R: CTAGAAGCACTTGCGGTGCA), Gapdh (F: TGC CAA GGC TGT GGG CAA GG, R: CCA GGC GGC ACG TCA GAT CC). Actb and Gaphd were used as housekeeping genes. The average of the Ct values of both housekeeping genes was used to calculate the relative expression levels for the respective genes of interest (Vandesompele et al. 2002 (link)).

Total RNA was harvested from miR- or siRNA-transfected cells and breast tissues using the ZR-Duet DNA/RNA MiniPrep kit (Zymo research, Irvine, CA, USA) according to the manufacturer’s recommendations. In order to quantify the levels of mature miR-7, the extracted RNA was reverse transcribed to cDNA using the miScript II RT Kit (Qiagen, Valencia, CA, USA). Afterwards, quantitative RT-PCR (qPCR) was performed with the miScript SYBR Green PCR Kit (Qiagen) and miScript Primer Assays as the primers. For the quantification of protein coding gene’s expression level, reverse transcription was carried out with ReverTra Ace qPCR RT Master Mix with gDNA remover (Toyobo, Japan) and PCR was performed with Kapa SYBR Fast qPCR Kit Master Mix ABI Prism (Kapa Biosystems, Inc., Wilmington, MA, USA). The reactions were assayed in triplicate on an ABI 7300 instrument (Applied Biosystems, Foster City, CA, USA). The expression of miR-7 and protein coding genes was normalized using endogenous U6 and GAPDH with the 2^−ΔΔCt calculation, respectively. The primers used for amplification of miRs and coding genes are listed in Supplementary Table S2.

Total RNAs were extracted using RNeasy (Qiagen). For quantitative reverse transcription PCR (RT-PCR), total RNA was reverse-transcribed into first-strand cDNA using ReverTra Ace (Toyobo) and an oligo(dT)20 primer. PCR amplification was performed using the KAPA SYBR FAST qPCR Master Mix ABI prism kit (KAPA Biosystems, Wilmington, USA) and StepOnePlus Real-Time PCR System (Thermo Fisher Scientific). The sequences of the PCR primers used are listed in Supplementary Table 4. The RNA expression levels of target genes were normalized to that of GAPDH or β-actin mRNA expression, and the results indicate the relative expression of the molecules.

cyiPSC-derived cartilage were frozen in liquid nitrogen and crushed by a Multi-Beads Shocker (Yasui Kikai, Japan). Total RNAs were extracted using RNeasy (Qiagen). For quantitative reverse transcription PCR (RT-PCR), 200 ng total RNA was reverse transcribed into first-strand cDNA using ReverTra Ace (Toyobo) and an oligo(dT)20 primer. The PCR amplification was performed using a KAPA SYBR FAST qPCR Master Mix ABI prism Kit (KAPA Biosystems, Wilmington) and StepOnePlus Real-Time PCR System (Thermo Fisher Scientific). Table 1 lists the PCR primers used.^{23 (link),24 (link)} The RNA expression levels were normalized to the level of GAPDH or ACTB expression, and the results indicate the relative expression of the molecules.