Rna extraction kit

Total RNA from homogenized tick samples was extracted using GE Healthcare RNA extraction kit (Chicago, USA). Complementary DNA (cDNA) was synthesized using a GoScript Reverse Transcriptase (Promega, Madison, Wisconsin, USA). Briefly, a mixture of 1 μl of RNA, 5 μl of poly T primers and 14 μl of nuclease-free water were preheated at 70°C for 10 min. The mixture was then placed on ice pending further steps. The enzyme mix (1 × first strand buffer, 4 μl of MgCl₂, 2 mM dNTP, 0.51 μl of SUPERase inhibitor, 1 μl of superscript II, 5 μl of the RNA mix, and RNase-free water) was then incubated in a thermocycler according to the manufacturer's cycling conditions; one cycle of 10 min at 25°C, 45 min at 37°C, 45 min at 42°C, followed by 15 min at 70°C. The cDNA was used immediately or stored at −20°C until it was needed for further analysis.

HAC culture pellets were collected and RNA was extracted using an RNA extraction kit (GE Healthcare) following the manufacturer’s protocol. A total RNA (0.25 μg) was converted to cDNA using a BIOLINE SensiFAST™ cDNA synthesis kit following the mixer and reaction protocol. Real-time PCR was performed to investigate gene expression using a BIOLINE SensiFAST™ SYBR® No-ROX Kit. The primers were purchased from Invitrogen (Table 1). The level of gene expression of the samples was compared with that of GADPH (the house-keeping gene) calculated using the 2^-ΔΔCT method [10 (link)].Table 1

Real-Time PCR Primers sequences

Gene	Real-time PCR primer sequence (5′-3′)
ACAN	Forward: ACTTCCGCTGGTCAGATGGAReverse: TCTCGTGCCAGATCATCACC
XT-1	Forward: GTGGATCCCGTCAATGTCATCReverse: GTGTGTGAATTCGGCAGTGG
XT-2	Forward: CGAATCGCCTACATCATGCTGGReverse: TAAACGGCCTTGAGGAGACG
CHSY-I	Forward: CCCGCCCCAGAAGAAGTCReverse: TCTCATAAACCATTCATACTTGTCCAA
ChPF	Forward: AGATCCAGGAGTTACAGTGGGAGATReverse: CCGGGCGGGATGGT
IL-1β	Forward: AAACAGATGAAGTGCTCCTTCCAGGReverse: TGGAGAACACCACTTGTTGCTCCA
MMP-13	Forward: TTGAGCTGGACTCATTGTCGReverse: GAGCCTCTCAGTCATGGAG
ADAMTs-4	Forward: CCCCAGACCCCGAAGAGCCAReverse: CCCGCTGCCAGGCACAGAAG
DEC	Forward: CCTGATGACCGCGACTTCGAGReverse: TTTGGCACTTTGTCCAGACCC
GADPH	Forward: GAAGGTGAAGGTCGGAGTCReverse: GAAGATGGTGATGGGATTTC

Total RNA from cultured scaffolds on days 7, 14, and 21 were extracted using an RNA extraction kit (GE Healthcare, UK), in accordance with the manufacturer’s protocol. 200 ng of total RNA were converted to cDNA using RevertAid™ H minus first strand cDNA synthesis kit (Fermentus Life science, USA). In order to determine the chondrogenic gene expression, SYBR Green detection was used, and the values were normalized using glyceraldehyde-3-phosphate dehydrogenase (GAPDH). A real-time quantitative polymerase chain reaction (PCR) was performed in a DNA Engine (ABi 7500) using SYBR GREENER qPCR UNIVERSAL (Invitrogen, USA). Primers sequences are as follows: Aggrecan core protein (ACAN) forward: 5′CTGTTCAGGGACAGAATGTGCT3′, Aggrecan core protein (ACAN) reverse: 5′TCGATATGCTTCACAGTTCTAGGG3′, Collagen type I (COL1A2) forward: 5′TTTTGGCCATCTCTTCCTTCA3′, Collagen type I (COL1A2) reverse: 5′TGTGGATGCCTC TTGGGTATC3′, Collagen type II (COL2A1) forward: 5′TCCTCTTCTTGAGCTGGACTCATTCT3′, Collagen type II (COL2A1) reverse: 5′CGCTCTGCAAACTGGAGGTC3′, SOX9 forward: 5′ GAAGGTGAAGGTCGGAGTC3′, SOX9 reverse: 5′GAAGATGGTGATGGGATTTC3′, GAPDH forward: 5′GAAGGTGAAGGTCGGAGTC3′, GAPDH reverse: 5′ GAAGATGGTGATGGGATTTC3′. Relative expression levels of each gene were calculated by normalizing them with the expression of GAPDH by the 2^-ΔCT method [56 (link)].