What is a good cDNA synthesis kit for downstream real time qPCR applications?

I am seeing a gene expression through Rtpcr .so wanted to know which method I should use for gene expression .is 2^-delta ct method I can use over 2^-delta delta ct

I have synthesised cDNA from 500ng/ul of RNA. The concentration of the cDNA Is about 5000ng/ul. Should I dilute my cDNA or just use it as it is for my qPCR? I usually use 2ul of cDNA for a 20ul reaction. Thank you!

These 2 analyze methods are recommended for specific gene expression analysis?

I am using sybr green mastermix and my cDNA quantity is okay. Still not getting amplification curve both for expected gene and internal control (GAPDH)

I had done PCR using GoTaq q PCR mastermix (Promega) for detection Hepatitis B virus cccDNA. For that purpose, which Ct value I will consider as detected or undetected?

I'm conducting a time course study on gene expression using RT-q PCR for samples treated with 4 conditions: vehicle, RA agonist, Calcitriol agonist or a combination of both agonists over 6 hours. I'm expecting to observe a gradual increase in expression over time for the combined treatment condition due to an additive effect of the ligands. Indeed, I have observed that for all of the time points except for the last one where the Ct value for my combined treatment is 30 while my untreated control at zero hour has a Ct value of around 28.85. Even the Ct value for the vehicle condition for my last time point is around 28.65 so, why am I getting such Ct for the combined treatment?

In-vivo biochemical assays like- BCA, Nitrite, GSH, Catalase, SOD, Ca+2 assay, LDH, and many more

Hi. I am having trouble interpreting the results of my experiment.

Chondrocytes were treated with LPS for 24 hours and then incubated with drugs that are expected to have anti-inflammatory effects and mRNA was extracted after 24 hours of incubation.

IL-6, TNF-a, and IL-1b mRNA expression levels were determined by qPCR. IL-6 was highest in the LPS(+)drug(-) group and decreased with increasing drug concentration.

However, TNF-a and IL-1b were expressed at levels similar to or lower than normal controls in the LPS(+)drug(-) group.

Comparatively, TNF-a and IL-1b were higher in the drug-treated group.

I'm curious that when I look at IL-6, it seems like the LPS signal is working, but I don't understand why TNF-a and IL-1b are lower in the LPS group. I'm wondering if this is possible.

If the mRNA peaked and then declined because of the longer time, would I need to reduce the LPS treatment time to get the desired results for TNF-a and IL-1b?

I used the ExoSpin kit to isolate EVs from Plasma using 0.22 double filtration and 10KDa Ultrafiltration device and resuspended in PBS (cacl and mg free) and still find that particles are clumping together.