Sybr green gene expression assay kit

Total RNA from articular cartilage tissues and chondrocytes was isolated with GenElute™ Total RNA Purification Kit (Sigma-Aldrich) and reverse transcribed into cDNA with miScript Reverse Transcription Kit (QIAGEN, Dusseldorf, Germany). TaqMan microRNA Assay Kit was used for miR-103a-3p and U6 expression analysis. SYBR Green Gene Expression Assay Kit (QIAGEN) was used to detect HMGB1 and β-actin expression. A quantitative real-time PCR assay was performed on the ABI7500 Instrument (Applied Biosystems, Warrington, UK). The primers used are listed as follows: miR-103a-3p (forward: 5’-ATCCAGTGCGTGTCGTG-3’; reverse: 5’-TGCTAGCAGCATTGTACAGG-3’); HMGB1 (forward: 5’-CCAACAGGCAAATGGGGTCT; reverse: 5’-TAACTGGTGGGCCAGGGATA-3’); U6 (forward: 5’-GCTTCGGCAGCACATATACTAAAAT-3’; reverse: 5’-CGCTTCACGAATTTGCGTGTCAT-3’) or β-actin (forward: 5’- CCAACCGCGAGAAGATGA-3’; reverse: 5’-CCAGAGGCGTACAGGGATAG-3’) was used as internal reference and the 2−ΔΔCt method was used to calculate the relative gene expression.

The influence
of LNPs on the hMSC gene expression was studied using a reverse transcription-polymerase
chain reaction using the real-time SYBR Green gene expression assay
kit (QIAGEN, Germany). The lignin- and LNP-exposed cells’ cDNA
was synthesized using the Fastlane Cell cDNA kit. The mRNA levels
of GSR, CAT, SOD1, GSTM3, GSTA4, and FAS genes were determined using
glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as the reference
gene. A total of 25 μL of the PCR reaction mix composed of 500
ng of template cDNA (2 μL), master mix (12.5 μL), RNase-free
water (8.5 μL), and primers (2 μL) was added per well.
After that, the plate was subjected to an RT-PCR machine for 40 cycles.^{33 (link)} Subsequently, the results were calculated by
a comparative threshold (C_t) method, and
fold changes were compared with the control. The mRNA level of expression
of the target genes was analyzed by the Athinarayanan et al. method.^{34 (link)} The ratio of the reference gene expression to
target gene expression levels was measured as ΔC_t = C_t (target genes) – C_t (GAPDH) and ΔΔC_t = ΔC_t (treated) –
ΔC_t (control), respectively. The
obtained data were used to plot the target gene expression using the
values of 2^−ΔΔC_t.

Total RNAs were extracted from tissue samples by using TRIzol reagent (Invitrogen, Carlsbad, USA), and then were reverse transcribed into cDNA with First Strand cDNA Synthesis Kit (Sigma-Aldrich).
TaqMan microRNA Assay Kit was used to analyze miR-582 and U6 expression. SYBR Green Gene Expression Assay Kit (QIAGEN)
was used to detect LIS1 and GAPDH expression. A quantitative real-time PCR assay was performed on the ABI7500 Instrument (Applied Biosystems, Warrington, UK).
Reaction conditions included: 95 °C for 30 s; 40 cycles of 95 °C for 5 s and 60 °C for 30 s. The primer sequences were listed as follows (Table 2).
The experiments were repeated at least three times. Results were analyzed by 2^-△△CT method.