To analyze gene expression, we harvested the NP cells/clusters at day 7. The total RNA was extracted from the NP cells/clusters using an RNeasy kit® (Qiagen). The samples were homogenized in guanidine isothiocyanate based proprietary component of the kit with 1% β‐mercaptoethanol (RLT buffer) with a handheld homogenizer pestle (Fisher) in accordance with the manufacturer's protocol. The samples were amplified with a reverse transcriptase (High Capacity cDNA Reverse Transcription Kit, Life Technology). Gene expression master mix and fluorescent‐labeled specific primers (TaqMan®, Life Technology) were mixed, followed by quantitative‐PCR (7900HT, Applied Biosystems, Foster City, CA). The TaqMan® primers were a collagen type‐II, COL‐2: Bt03251837_mH; aggrecan core protein, AGG: Bt03212189_m1; proliferating cell nuclear antigen, PCNA: Bt03211154_g1; chondroitin sulfate N‐acetylgalactosaminyltransferase 1, CSGALNACT1: Bt03272948_m1, matrix metalloproteinase‐13, MMP‐13: Bt03214051_m1; and glyceraldehyde 3‐phosphate dehydrogenase, GAPDH: Hs03929097_g1 (Life Technology, Carlsbad, CA). Expression Suite Software v1.0.4 was used to analyze the data.
Handheld homogenizer pestle
Manufactured by Thermo Fisher Scientific
The Handheld homogenizer pestle is a laboratory instrument used for the mechanical disruption and homogenization of small samples. It is designed to efficiently mix and blend samples within test tubes or small containers.
Automatically generated - may contain errors
2 protocols using handheld homogenizer pestle
1
Quantifying Gene Expression in Bovine Nucleus Pulposus Cells
2
Quantifying NP Cell Gene Expression
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To analyze gene expression, we harvested the NP cells/clusters on defined days. The total RNA was extracted from the NP cells/clusters using an RNeasy kit (Qiagen, Germantown, Maryland). The samples were homogenized in the guanidine isothiocyanate‐based proprietary component of the kit with 1% β‐mercaptoethanol (RLT buffer) using a handheld homogenizer pestle (Fisher Scientific) in accordance with the manufacturer's protocol. The samples were amplified with a reverse transcriptase (High Capacity cDNA Reverse Transcription Kit, Thermo Fisher). A gene expression master mix and fluorescent‐labeled specific primers (TaqMan, Thermo Fisher) were mixed, followed by qPCR (QuanStudio version 1.4, Applied Biosystems, Foster City, California). We measured the expression of the typical molecules aggrecan core protein (Acan) and collagen type‐II (Col2a1) and the minor molecule collagen type‐I (Col1a1) in the ECM of NP as well as a catabolic molecule in the NP matrix metalloproteinase‐13 (Mmp‐13). All expression levels were compared with an intrinsic control, glyceraldehyde 3‐phosphate dehydrogenase (GAPDH), in each experiment. The TaqMan primers were a Col1a1: Bt03225358_g1; Col2a1: Bt03251837_mH; Acan: Bt03212189_m1; Mmp‐13: Bt03214051_m1; and GAPDH: Bt03210919_g1 (Life Technology, Carlsbad, California). Expression Suite Software v1.0.4 was used to analyze the data.
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