(C) Limit of detection of ten replicates of the two test formats. (D) Standard curve generated by plotting the number of IVT RNA copies (x-axis) vs. the mean of the corresponding RT-PCR threshold cycle (Ct) value (y-axis) of three independent experiments (Original gel images in Fig....
Mouse OPN4S and OPN4L share the first 454 amino acids but differ in their C-terminal tails17. Quantitative RT-PCR indicates thatOpn4SmRNA level is 40 times that ofOpn4Lin adult mice, and immunostaining with OPN4S- and OPN4L-specific antibodies indicates that both protein isoforms are present...
Total RNA from the PC cell lines was extracted using a ZEB kit (Ebioscience). RNA was used for cDNA synthesis with the Superscript III Reverse Transcription Reagent (Life Technologies). The real-time PCR reaction was performed according to the protocol of the SYBR Premix Ex Taq kit (Takara)...
RNA-seq and small RNA-seq are powerful, quantitative tools to study gene regulation and function. Common high-throughput sequencing methods rely on polymerase chain reaction (PCR) to expand the starting material, but not every molecule amplifies equally,
annealing for 15-60 s at 40-65°C, and extension for 30-120 s at 72°C, with a final extension at 72°C for 5 min. Specific conditions varied across loci and taxa. The PCR products were subsequently desalted and concentrated using an ArrayIt®PCR Product Purification Kit (TeleChem Int...
vs. Phycoerythrin (PE-A). P2 and P3 demarcate cells sorted as GFP- and GPP+, respectively. (C) Quantitative RT-PCR analysis (qRT-PCR) was performed of RNA derived from GFP+ and GFP- cells from tails of 36 hpf embryos. Real time PCR ratio was determined by normalization toβ-actin(...
After second-strand synthesis, PCR amplification was performed using KAPA HiFi PCR Mix (Kapa Biosystems KK2602). Specifically, a 40 μL PCR Mastermix consisting of 25 μL of KAPA 5X Mastermix, 0.4 μL of 100 μM ISPCR oligo, and 14.6 μL of nuclease-free water was combined with 2,00...
(D and E) qRT-PCR analyses of Kdm4b, Mbp, Plp, Myrf, Mag, and Mog in siKdm4b (D) or siPrkcq-treated (E) rat oligodendrocytes. Data are presented as mean ± SEM.; n = 3 independent experiments; two-tailed unpaired Student’s t test. (F) Examples of cage activity rhythms record...
*Best practice genotyping should be PCR-based sequence analysis along with MLPA as addressed in 6.1. of the manuscript. **Second pathogenic variant in trans not detected. Full size image It is still controversial whether an ACTH-stimulation test should be performed as the first test for carrier...
RNA extraction and quantitative reverse transcription PCR After perfusion of the heart with ice-cold PBS, the mouse brain was collected. The cortex then was isolated and grinded in 1 mL of TRIzol on ice, and RNA was extracted using TRIzol reagent following the protocols supplied by the manufa...