Nana drop 2000

Total RNA was extracted from ovary or skeletal muscle tissues using a RNApure total RNA isolation kit (Bio Teke, Beijing, China). The concentration of RNA was detected on an ultraviolet spectrophotometer (Nana Drop 2000, Thermo Scientific, USA). Subsequently, cDNA was synthesized using M-MLV Reverse Transcriptase (Takara, Japan). Real time PCR was carried out using Taq HS Perfect Mix (Takara) and SYBR Green (Bio Teke) on an Exicycler™ 96 Real-Time Quantitative Thermal Block ⁽BIONEER, Korea). The primer sequences are presented in Table 1. The mRNA expression level was calculated by 2^-△△CT method.
Table 1

Oligonucleotide primer sets for real-time PCR

Name	Sequence (5′¬3′)	Length
CYP17 F	TGGAGGTGATAAAGGGTT	18
CYP17 R	CGTCAGGCTGGAGATAGA	18
CYP19 F	GCCTGTCGTGGACTTGGT	18
CYP19 R	TAAATTCATTGGGCTTGG	18
PPARγ F	TACCACGGTTGATTTCTC	18
PPARγ R	AATAATAAGGCGGGGACG	18
PGC-1α F	GAACAAGACTATTGAGCGAACC	22
PGC-1α R	GAGTGGCTGCCTTGGGTA	18
β-actin F	CCACTGCCGCATCCTCTT	18
β-actin R	GGTCTTTACGGATGTCAACG	20

B. bigemina was cultured under ambient oxygen (approx. 20% O₂ and 5% CO₂) or low oxygen (3% O₂, 5% CO₂, and 92% N₂) conditions. Samples were collected until parasitemia reached 5%. RNA was extracted by using Trizol (Invitrogen, Waltham, MA, USA). RNA purity and concentration were monitored by using a Nanadrop 2000 (Thermofisher, Waltham, MA, USA). Five hundred nanograms of RNA was used for cDNA synthesis by using the SuperScript™ First-Strand kit (Invitrogen, Waltham, MA, USA).
Three pairs of primers were designed based on the sequence of BbigLDH and 18S rRNA by using RT-PCR design wizard in Clone manager version 8, respectively. All the primers were subjected to quantitative polymerase chain reaction (qPCR); the pairs showed stable results, and the melt curve was selected for further study. The LDH transcription level was determined by qPCR, and the B. bigemina 18S rRNA gene was used as a reference. The primers for qPCR are listed in Table 1.

The total RNA was isolated using RNAsimple Total RNA Kit (TIANGEN DP419), and the RNA concentration was measured using NanaDrop 2000 (Thermo Scientific). The integrity of RNA was tested by running AGE (agarose gel electrophoresis). Reverse transcription of RNA utilized PrimeScript^™ RT Master Mix (TAKARA RR036A), subsequently followed by real-time PCR using SYBR^®Premix Ex Taq^™ (TAKARA RR420A). Relative abundance of mRNA transcript for the selected genes was calculated using the ΔΔCt approximation relative to housekeeping gene GAPDH. The real-time PCR primers were MYCN-F 5′-ACAGTGAGCGTCGCAGAAAC-3′, MYCN-R 5′-AGCAAGTCCGAGCGTGTTC-3′, GAPDH-F 5′-AATCCCATCACCATCTTCC-3′, and GAPDH-R 5′-CATCACGCCACAGTTTCC-3′.