Maxwell 16 cell lev total rna purification kit

Samples for gene expression assay were collected on Days 7 and 14. After being immediately snap‐frozen in liquid nitrogen, the samples were stored at −80°C. Total RNA was extracted using a Maxwell® 16 Cell LEV Total RNA Purification Kit (AS1280; Promega, USA) in accordance with the manufacturer's protocol. Reverse transcription was undertaken using a High‐Capacity cDNA reverse Transcription Kit (4368813; Applied Biosystems, USA). RT‐qPCR was performed with the StepOne™ real‐time PCR system (4376357; Applied Biosystems) with TaqMan® Universal Master Mix (4352042; Applied Biosystems). The amplification was performed as follows: initial activation of polymerase at 95°C for 20 s followed by 40 cycles of PCR, at 95°C for 1 s (denature) and 60°C for 20 s (anneal and extend).
The tailored panels of gene expression assay for cytoskeletal rearrangement and osteogenesis were designed with reference to the predetermined TaqMan® gene expression arrays for focal adhesion (4413255‐RPMFWWX; Applied Biosystems), cytoskeleton regulators (4413255‐RPPRJ2T; Applied Biosystems), and osteogenesis (4413255‐RPZTD3T; Applied Biosystems). A set of primers used in the study is listed in Tables [Link], [Link]. Relative expression of each mRNA was calculated with the ΔΔCt method.⁴¹

RNA was extracted from sections of frozen mouse brain cerebrum representing each treatment group (n = 6 per group) using the Maxwell 16 LEV simplyRNA Tissue Kit (Promega, Ipswich, MA, USA; AS1270). RNA was extracted from cultured cells using the Maxwell 16 Cell LEV Total RNA Purification Kit (Promega; AS1225). Corresponding cDNA was prepared using the SuperScript III First-Strand Synthesis System (Invitrogen).
Commercially available TaqMan assays (S2 Table) for droplet digital PCR were prepared using the One-Step RT-ddPCR Advanced Kit for Probes (Bio-Rad, Hercules, CA, USA; 1864022). Each 20-μL reaction mix consisted of 10 μL ddPCR Supermix for probes (no dUTP) 1 μL of each target probe, 1 μL of reference probe and up to 8 μL of cDNA or water, making a final volume of 13 μL. The reaction mix was then partitioned into droplets using a QX100 Droplet Generator (Bio-Rad). Target and reference probe fluorescence amplitudes for each droplet were analyzed using a QX200 Droplet Reader (Bio-Rad) for digital absolute quantification. Each sample was analyzed in duplicate.

Samples for gene expression assay were snap-frozen in liquid nitrogen and stored at −80°C. Total RNA was extracted using a Maxwell^® 16 Cell LEV Total RNA Purification Kit (AS1280; Promega, United States) in accordance with the manufacturer’s protocol. Reverse transcription was then undertaken using a High-Capacity cDNA reverse Transcription Kit (4368814; Applied Biosystems, United States). RT-qPCR was performed with the StepOne™ real-time PCR system (4376357, Applied Biosystems, United States) with TaqMan™ Gene Expression Assay (4331182, Thermo Fisher Scientific, United States). The primers used were Runt-related transcription factor 2 (RUNX2, Rn01512298_m1, Thermo Fisher Scientific, United States), Osterix (Rn01761789 m1, Thermo Fisher Scientific, United States), and Glyceraldehyde-3-phosphate dehydrogenase (GAPDH, Rn01749022 g, Thermo Fisher Scientific, United States). The amplification was performed as follows: initial denaturation at 95°C for 20 s followed by 40 cycles at 95°C for 1 s and 60°C for 20 s. Relative gene expression was calculated by the ΔΔCt method, normalized by the endogenous control, GAPDH (Livak and Schmittgen, 2001 (link)). The data are presented as a mean value ± standard error (s.e.m) of three replicates.