Human gingival biopsies were taken from subjects with healthy periodontium and chronic periodontal disease according to the AAP/ADA classification64 . All enrolled participants into this study provided written informed consent, which was approved by the Institutional Review Board (IRB) of the University of North Carolina at Chapel Hill (IRB number, 15–0335). Upon removal, total RNA was isolated from these human gingival tissue biopsies using the Qiagen RNA extraction kit and cDNA was synthesized using Vilo according to the manufacturer’s instructions. Expression level of 5 different isoform of IL-37 were measured by qPCR using the ABI 7500 real-time RCR system with SYBR green (Applied Biosystems). Isoform specific primers listed below were designed by flanking the junction of exon and confirmed by Agarose gel. Isoform1: Forward: 5-TCACACAAGTCCAAAGGTGA-3, Reverse: 5- AGCCAGCTTCATCAGTTTCT-3. Isoform2: Forward: 5-GCTTAGAAGGTCCAAAGGTGAA-3, Reverse: 5- GAGCTCAAGGATGAGGCTAATG-3. Isoform3: Forward: 5- GCTGCTTAGAAGAGATCTTCTTT-3, Reverse: 5- CTGAAGGGATGGATGACTTTG-3. Isoform4: Forward: 5-TCACACAAAGATCTTCTTTGCA-3, 5-CAGCCAGCTTCATCAGTTTC-3. Isoform5: Forward: 5-GCGCTTAAGAGGTCCAAAGGT-3, 5-GCTATGAGATTCCCAGAGTCCAG-3.
Abi 7500 real time rcr system
Manufactured by Thermo Fisher Scientific
The ABI 7500 real-time PCR system is a laboratory instrument used for quantitative real-time polymerase chain reaction (qRT-PCR) analysis. It is capable of performing sensitive and precise detection and quantification of nucleic acid sequences.
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2 protocols using abi 7500 real time rcr system
1
Quantifying IL-37 Isoform Expression in Gingival Tissues
2
Quantitative RT-PCR Analysis of ASMC Markers
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Total RNA was extracted from ASMCs using TRIzol® reagent (Invitrogen; Thermo Fisher Scientific, Inc.) and reverse transcribed into cDNA using the PrimeScript RT Reagent kit (Takara Bio, Inc., Otsu, Japan), according to the manufacturer's protocol. The RT-qPCR reactions were performed using ABI7500 real-time RCR System (Applied Biosystems; Thermo Fisher Scientific, Inc.). SYBR-Green Master Mix kit and the following thermocycling conditions were used for qPCR: 30 cycles of 30 sec at 94°C, 30 sec at 55°C and 30 sec at 72°C. The following primer sequences were used for PCR: MMP-9 forward, 5′-TCATCCAGTTTGGTGTCGC-3′ and reverse, 5′-AGTGGGCATCTCCCTGAAT-3′; TIMP1 forward, 5′-GCCTCTGGCATCCTCTTG-3′ and reverse, 5′-CTGCGGTTCTGGGACTTG-3′; transforming growth factor β-1 (TGFβ1) forward, 5′-AGGCGGTGCTCGCTTTG-3′ and reverse, 5′-TGCGTTGTTGCGGTCCA-3′; fibroblast growth factor 1 (FGF-1) forward, 5′-GATGGCACAGTGGATGGGAC-3′ and reverse, 5′-AAGCCCGTCGGTGTCCATGG-3′; and GAPDH forward, 5′-CCTCGTCTCATAGACAAGATGGT-3′ and reverse, 5′-GGGTAGAGTCATACTGGAACATG-3′. The fold-change in the expression of each target mRNA relative to GAPDH was calculated using the 2−ΔΔCq method (32 (link)).
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