Lightcycler real time pcr machine

Total RNA was isolated from cells with Trizol reagent (Takara, Shiga, Japan) and RNeasy^® Mini Kit (QIAGEN, Hilden, German), according to the manufacturer’s instruction. cDNA was synthesized from 1 μg of total RNA using an iScript™ cDNA Synthesis Kit (Bio-Rad Laboratories Inc., Hercules, CA, USA). Then, the qRT-PCR analysis was performed with FastStart Essential DNA Green Master with LightCycler Real-Time PCR machine (Roche, Basel, Switzerland). The primer sequences used in qRT-PCR analysis were shown below.
Filaggrin forward 5′- AGT GCA CTC AGG GGG CTC ACA -3′ and Filaggrin reverse 5′- CCG GCT TGG CCG TAA TGT GT -3′; Involucrin forward 5′- TTG GTC AGT GAA GCG ATG AG -3′ and Involucrin reverse 5′- AGA TCT GTC TGC AGG GCT GT -3′; Loricrin forward 5′- TCA TAA GAA ACC CCG CTG AG -3′ and Loricrin reverse 5′- AAG GAA GGA GAG CCT GGA AG -3′; Caspase-14 forward 5′- CAA ACA CAT GGG TCA CTT GC -3′ and Caspase-14 reverse 5′- CAG AAC TGC TGA GCC TAC CC -3′; GAPDH forward 5′- GGA GCG AGA TCC CTC CAA AAT -3′ and GAPDH reverse 5′- GGC TGT TGT CAT ACT TCT CAT GG -3′. All results were normalized to internal control.

In order to enrich bone marrow for hematopoietic stem and progenitor cells, c-Kit⁺ cells were isolated by magnetic separation with anti-c-Kit labeled magnetic microbeads (Miltenyi Biotec, Bergisch Gladbach, Germany), following the instructions provided by the manufacturer. The number of c-Kit⁺ cells was determined and RNA was subsequently isolated using a RNAeasy mini kit (Qiagen, Solna, Sweden). Analysis of gene expression was performed with one- step real-time reverse transcription PCR using QuantiTect™ SYBR® Green RT-PCR kit (Qiagen). The following PCR conditions were used; reverse transcription at 50°C for 20 minutes, inactivation at 95°C for 15 minutes, 50 cycles of denaturation at 94°C for 15 s, annealing for 25 s at 60°C, and extension at 72°C for 15 s. All primer sequences can be provided upon request. The PCR reactions were all run a LightCycler™ real- time PCR machine (Roche Diagnostics, Basel, Switzerland). The Cycle threshold (C_T) values were estimated with the LightCycler Software v 4.1 and transcript levels were normalized by subtracting the corresponding β –actin values. Control was set at one differences and presented as 2^-ΔKOCt-WTCt.

Cells‐to‐CT kit (Thermo Fisher Scientific) was used to extract total RNA from OVCA cells in vitro. Total RNA was extracted from tumor tissue using RNeasy Kit (Qiagen) and cDNA was prepared from 100 ng total RNA using Superscript III First Strand synthesis kit and oligo‐dT primers (Thermo Fisher Scientific). mRNA expressions were quantitated using CHOP (Hs01090850_m1), XBP1s (Hs03929085_g1), ATF3 (Hs00910173_m1), ATF4 (Hs00909569_g1), and DR5 (Hs00366278_m1) transcripts from Taqman probes (Life Technologies) in a LightCycler real‐time PCR machine (Roche Diagnostics, Indianapolis, IN). qPCR results were presented as means from three independent experiments and normalized to GAPDH (Hs02786624_g1) transcript. The comparative C_t formula was used in fold change calculations.

The blood clots remaining in the calvarium defects were collected as described above. The total RNA from the blood clot samples was extracted with the TRIzol reagent (Beyotime Biotechnology, China). Relative mRNA expression levels of immune-system-related factors, bone remodeling-related factors, fibrogenesis-related factors, and angiogenesis-related factors were analyzed by reverse-transcription quantitative PCR (RT-qPCR). RT-qPCR primers used in this study are listed in Supplementary Table 1. SYBR Premix Ex Taq™ (Takara, Japan) was employed for assays on a Light Cycler Real Time PCR machine (Roche, Germany). The average values of three independent tests for each gene were normalized by Z-score after being taken the logarithm of 2. Then clustering heatmap was made by MeV software (Multi Experiment Viewer, version 4.9.0, http://www.tm4.org/mev.html). The interaction of these factors was analyzed on the STRING website (https://string-db.org/cgi/input.pl?sessionId=Cuzp3bpg46U5&input_page_active_form=multiple_identifiers).