Rotor gene q 2plex hrm platform

Gene expression studies of targeted endogenous cellular antioxidant enzymes were performed through qRT-PCR approach. A one-step qRT-PCR kit (QuantiFast SYBR Green RT-PCR Kit, Qiagen) was used to quantify the RNA targets. A total of 20 ng of RNA sample (final amount per reaction tube = 2 ng) was mixed with reagent kits and oligonucleotide primer sets as per manufacturer's instructions. The primers used in this experiment were as follows: 

Catalase (CAT)

 

(Gene ID: 24248)

 

Species: Rattus norvegicus

 

Forward Primer:

 

5′-CGCCTGTGTGAGAACATTGC-3′

 

Reverse Primer:

 

5′-TAGTCAGGGTGGACGTCAGT-3′

 

Glyceraldehyde 3-phosphate dehydrogenase (GAPDH)

 

(Gene ID: 24383)

 

Species: Rattus norvegicus

 

Forward Primer:

 

5′-CAG GGC TGC CTT CTC TTG TG-3′

 

Reverse Primer:

 

5′-CTT GCC GTG GGT AGA GTC AT-3′

Amplification reactions of RNA targets were performed via Rotor-Gene Q 2plex HRM Platform (Qiagen). Settings for reaction cycles were configured as specified in the kit manual. All reactions were normalized to mRNA expression of the housekeeping gene, GAPDH for Rattus norvegicus. All samples were prepared in triplicate and relative gene expression levels of RNA targets were normalized to that of negative control cells.

To validate RNA sequencing results, selected orthologous pair genes were used for qPCR analysis with three biological replicates of CF, WK, and F1. Total RNA (1 μg) was used for reverse transcription with Superscript III Reverse transcriptase (Invitrogen, USA) and 1 μL of 1/20 diluted cDNA was used for a PCR reaction. Primers were listed in Table S5. qPCR was performed for 10 min of denaturation at 95 °C following 40 cycles of 10 s at 95 °C, 15 s at 60 °C, and 35 s at 72 °C with Rotor-Gene Q 2plex HRM platform and QuantiFast SYBR Green PCR kit (Qiagen, Germany).

Total RNA was extracted by using TRIzol reagent (Life Technologies, Carlsbad, CA, USA). The purity and quality of RNA were confirmed by defining the ratio of absorbance at 260 and 280 nm wavelengths (NanoDrop^® ND-1000, Thermo Scientific, Waltham, MA, USA). A sample of 3 µg total RNA was reverse transcribed into cDNA. For mRNA measurement, diluted cDNA was amplified using the Rotor-Gene SYBR Green PCR Kit (Qiagen, Hilden, Germany) in a Rotor-Gene Q 2plex HRM Platform (Qiagen). Reaction conditions were carried out for 35–40 cycles (5 min at 95 °C, 5 s at 95 °C and 10 s at 60 °C). All procedures for RNA extraction and qPCR have been described previously [57 (link),58 (link),59 (link)].
The primers were designed using reported cDNA sequences: 1.TNF-α, Forward 5′-CTC TTC TCA TTC CCG CTC GTG-3′ and Reverse 5′-GGA ACT TCT CCT CCT TGT TGG G-3′; 2.IL-1β, Forward 5′-GTT TGA GTC TGC ACA GTT CCC-3′ and Reverse 5′-CAA CTA TGT CCC GAC CAT TGC-3′; 3.IL-6, Forward 5′-TTC TTG GGA CTG ATG TTG TTG AC-3′ and Reverse 5′-AAT TAA GCC TCC GAC TTG TGA AG-3′; 4. β-actin, Forward 5′-GAC CCA GAT CAT GTT TGA GAC CTT C-3′and Reverse 5′-GAG TCC ATC ACA ATG CCW GTG G-3′.