siRNA / miRNA gene silencing Rat MTLn3 (rat mammary adenocarcinoma breast cancer cell line)

RNA-Seq is a method to sequence RNA by applying Next Generation Sequencing (NGS). The quality of RNA is critical for the success of RNA-Seq. The integrity of RNA is measured by the RNA integrity number (RIN). RIN is computed from RNA electrophoresis and electropherogram profiles (the peak area of the 28S rRNA should be approximately twice the peak area of the 18S rRNA). If you get the RIN value lower than 7, the possibility of getting the low quality of RNA-seq data is high. To get a high quality RNA, it is better to work with fresh samples or snap-freeze the tissues in liquid nitrogen as quickly as possible and store them at -80°C until further use. Make sure designated areas and all your filter tips, microfuge tubes, plastic, and glassware are RNase-free.

Get tips on using ON-TARGETplus Mouse Sirt2 (64383) siRNA - SMARTpool to perform siRNA / miRNA gene silencing Mouse - RAW264.7 Sirt2

Get tips on using ON-TARGETplus Mouse Bnip3 (12176) siRNA - SMARTpool to perform siRNA / miRNA gene silencing Mouse - RAW264.7 BNIP3

Get tips on using ON-TARGETplus Mouse Rheb (19744) siRNA - SMARTpool to perform siRNA / miRNA gene silencing Mouse - RAW264.7 Rheb

Get tips on using Human/Mouse/Rat Total HSP70/HSPA1A DuoSet IC ELISA to perform ELISA Rat - HSP70

Get tips on using Arp2 siRNA (h) to perform siRNA / miRNA gene silencing Human - T47-D Arp-2

Get tips on using siRNA TICAM-1 to perform siRNA / miRNA gene silencing Human - HT-29 TICAM-1

ELISA is the most commonly used method of detecting and quantifying the concentration of an antigen in an unknown sample. During the experiment, If you get a weak signal, then make sure reagents are at room temperature before starting the assay. Try increasing incubation times to ensure maximal antibody binding and amplify the signal. Secondly, if you get values above 0 in the negative control indicates a high background signal. Try to consider reducing your antibody concentration and prevent non-specific binding of antibodies by using affinity-purified antibody and suitable blocking buffers. To avoid high well to well variation, do not stack plates during incubation, no bubbles in the plate and wash wells thoroughly to avoid variation.

ELISA is the most commonly used method of detecting and quantifying the concentration of an antigen in an unknown sample. During the experiment, If you get a weak signal, then make sure reagents are at room temperature before starting the assay. Try increasing incubation times to ensure maximal antibody binding and amplify the signal. Secondly, if you get values above 0 in the negative control indicates a high background signal. Try to consider reducing your antibody concentration and prevent non-specific binding of antibodies by using affinity-purified antibody and suitable blocking buffers. To avoid high well to well variation, do not stack plates during incubation, no bubbles in the plate and wash wells thoroughly to avoid variation.

As autophagy is a multi-step process which includes not just the formation of autophagosomes, but most importantly, flux through the entire system, including the degradation upon fusion with lysosomes, which makes it quite challenging for detection. There are several methods for detection in mammalian cells, including immunoblotting analysis of LC3 and p62 and detection of autophagosome formation/maturation by fluorescence microscopy, Currently, there is no single “gold standard” for determining the autophagic activity that is applicable in every experimental context, hence it is recommended to go for the combined use of multiple methods to accurately assess the autophagic activity in any given biological setting.