Sapphireamp fast pcr master mix

To determine the expression of phorbol-12-myristate-13-acetate -induced protein 1 (Noxa), total RNA was isolated by chomczynski-fenol method [17 (link)] from HUVECs incubated in absence (control) or presence of high d-glucose (25 mmol/L, 24 h), H₂O₂ (100 µM) and/or G. tinctoria extract (200 µg/L) or vehicle (DMSO, 1% v/v). Non-quantitative polymerase chain reaction (PCR) was performed using Swift Max Pro thermal cycler (Esco technologies, Horsham, USA) in a reaction mixture containing 0.5 μmol/L primers, deoxynucleotide triphosphates, thermostable deoxyribonucleic acid polymerase and a reaction buffer (SapphireAmp Fast PCR Master Mix, Clontech laboratories, Mountain View, USA). The oligonucleotide primers for Noxa were gently provided by Dr. Roxana Pincheira (University of Concepción) and 28S was used as housekeeping. The amplicon images (PCR bands) in agarose gel were captured under ultraviolet (UV) light, documented and analyzed using ImageJ software (Java-based imaging processing program, National Institute of Health, USA) [17 (link)].

DNA was extracted from bacterial colonies with the QuickExtract™ Bacterial DNA Extraction Kit (Waltham, MA, USA) or the mBio UltraClean^® Microbial DNA Isolation Kit. Genomic DNA (Carlsbad, CA, USA) was used to amplify 16S rRNA genes using the primers 27F (5’-AGAGTTTGATCMTGGCTCAG-3’) and 1492R (5’-GGTTACCTTGTTACGACTT-3’) [36 (link)] with the SapphireAmp FAST PCR master mix (Clontech/Takara, Mountain View, CA, USA) for PCR. Two Gram-variable bacilli designated A4 and A15 had the most unique 16S rRNA sequences that were <94% similar to previously identified Sporosarcina spp. Genomic DNA was sent to the Bioinformatics Center at the Oregon State University, Center for Genome Research and Biocomputing (https://cgrb.oregonstate.edu/ (accessed on 18 April 2019), Corvallis campus, for whole genome sequencing using Illumina’s MiSeq platform 150 bp paired-end sequencing chemistry. Raw fastq files were used for de novo genome assembly with SPAdes v 3.10.1 [37 (link)]. Assembled scaffolds of strains A4 and A15 genome sequences were submitted to NCBI for BLAST analyses using Microbial genome Blast analysis tools [38 (link)]. Genome annotation was completed using the IMG/MER Pipeline [39 (link)]. Conserved domain analyses [40 (link)] and clusters of orthologous genes (COG) profiles [41 (link)] were also completed for both the A4 and A15 genomes.

Cells were collected when LCL cultures reached 30mL, and fibroblasts were ∼80% confluent in three T175 plates. All cell counting was performed using the Countess II cell counter (Life Technologies) and Trypan Blue exclusion. Cultures with >85% cell viability were used in subsequent experiments. To preserve cells for subsequent RNA extraction, 1 million cells were washed in PBS, pelleted, and resuspended in 500 μL TRIzol (Invitrogen) or 200 μL RNAprotect Cell Reagent (Qiagen). Cell suspensions were then frozen at −80°C. Cell cultures were maintained at low passage number; RNA-sequencing experiments were performed on samples at or below passage 4.
Periodically, and on each passage used for experiments, cell cultures were confirmed negative for mycoplasma contamination using either the MycoAlert Kit (Lonza) following the manufacturer’s instructions, or PCR using SapphireAmp Fast PCR Master Mix (Takara) and the following primers:
Myco2(cb): 5′ CTTCWTCGACTTYCAGACCCAAGGCAT-3′
Myco11(cb): 5′ ACACCATGGGAGYTGGTAAT-3′
PCR for GAPDH was performed on the same sample, using the following primers:
hGAPDH-F: TGT CGC TGT TGA AGT CAG AGG AGA
hGAPDH-R: AGA ACA TCA TCC CTG CCT CTA CTG.
Known mycoplasma positive and negative samples were used as a reference.