Hot firepol evagreen qpcr mix plus with rox

Cells were lysed in TRI Reagent (Sigma) and RNA was isolated following the manufacturer’s protocol. RNA concentration and quality were measured with a ND-1000 (NanoDrop) spectrometer. cDNA was synthesised from 500 ng of total RNA using High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems) and quantified with the 5x HOT FIREPol® EvaGreen® qPCR Mix Plus with ROX (Solis BioDyne) using a Rotor-Gene Q cycler (Qiagen) and the respective primer pairs (Table 1). Expression values were normalised to GAPDH mRNA.Table 1

Sequences of RT-qPCR primers

Gene name	Sense primer	Anti-sense primer
GAPDH	CGACCACTTTGTCAAGCTCA	TGTGAGGAGGGGAGATTCAG
PDPN	GCATCGAGGATCTGCCAACT	CCCTTCAGCTCTTTAGGGCG
NTN1	TGCCATTACTGCAAGGAGGG	TTGCAGGTGATACCCGTCAC
PPP1R14A	GTGGAGAAGTGGATCGACGG	CCCTGGATTTTCCGGCTTCT
A2M	AGAGCAGCATAAAGCCCAGT	TCTCAGTGGTCTCAGTGTGGA
p21	GGCGGCAGACCAGCATGACAGATT	GCAGGGGGCGGCCAGGGTAT
SNEV	TCATTGCCCGTCTCACCAAG	GGCACAGTCTTCCCTCTCTTC
FGF7	TGGCAATCAAAGGGGTGGAA	GCCATAGGAAGAAAGTGGGCT
CCL2	GAAAGTCTCTGCCGCCCTTC	ACAGATCTCCTTGGCCACAA
CXCL1	TCAATCCTGCATCCCCCATAG	CAGGAACAGCCACCAGTGAG
CXCL8	CTCTTGGCAGCCTTCCTGATTT	ACAGAGCTCTCTTCCATCAGA
IL11	ATGAACTGTGTTTGCCGCCT	GGGAATCCAGGTTGTGGTCC

Cells were lysed in TRI Reagent (Sigma, St. Louis, MO, USA), and RNA was isolated following the manufacturer’s protocol. RNA concentration and quality were measured with a ND-1000 NanoDrop spectrometer. cDNA was synthesized from 500 ng of total RNA using High-Capacity cDNA Reverse Transcription Kit (ThermoFisher, MA, USA) and quantified with 5x HOT FIREPol^® EvaGreen^® qPCR Mix Plus with ROX (Solis BioDyne, Tartu, Estonia) using a Rotor-Gene Q cycler (Qiagen, Hilden, Germany). Expression values were normalized to GAPDH mRNA.

Mammary gland tissues were taken by biopsy gun at prenatal, milking and drying periods as illustrated previously (26 (link)). Samples were kept at –80°C until RNA extraction. Total RNAs were extracted from the samples with RNX plus (Cinna Clon Inc., Iran) and the cDNAs were synthesized from 2 μg of total RNA using RT-PCR kit (Vivantis, Malaysia).
To measure the gene expression of the BLG and CSN1S1, real-time PCR reaction was used (28 (link),29 (link)). Real-time PCR reaction was carried out in a total volume of 20 μL including cDNA, 5X HOT FIREPol® EvaGreen®qPCR Mix Plus with ROX (Solis BioDyne, Estonia), related forward and reverse primers and distilled water, by using a Miniopticon real-time PCR system (Bio-Rad Laboratories, USA).
Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), was used as the reference gene (30 (link)-32 (link)).
The oligonucleotide sequences of the primers for the candidate genes were as follows: CSN1S1: 5′-CACAGTATGAAAGAGGGAAAC, 5′-ATGGGATTAGG GATGTCAGAG; BLG: 5′-GACTTGGTACTCCTTGGCTAT, 5′-TTGAACACCGCAGGGATCTTG; GAPDH: 5′- AGTCAAGGCAGAGAACGGGAA, 5′-ACAAACATG GGGGCATCAGCA. Real-time PCR reactions were performed as described previously (26 (link)).

Cells were lysed in TRI Reagent (Sigma-Aldrich, Missouri, United States), and RNA was isolated following the manufacturer’s protocol. RNA quality and concentration were measured with a NanoDrop One UV–Vis Spectrophotometer (Thermo Scientific, Massachusetts, United States). cDNA was synthesized from 500 ng of total RNA with the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, California, United States). Quantification was conducted with the 5x HOT FIREPol^® EvaGreen^® qPCR Mix Plus with ROX (Solis BioDyne, Estonia) using the Rotor-Gene Q (QIAGEN, Netherlands) and the respective primer pairs (Table 1). Expression values were normalized to GAPDH mRNA.