Total RNA was purified from mouse tibialis anterior, soleus, triceps, quadriceps, latissimus dorsi, diaphragm, heart, and brain after flash freezing in liquid nitrogen. The isolation process started with a crude homogenization of 25 mg of tissue in proteinase K solution (10 mM Tris-Cl, pH 7.5 [Wisent]; 10 mM EDTA [Sigma-Aldrich]; 2% SDS [Roche]; 500 mM NaCl [Fisher Scientific]; 1.5 mM MgCl2 [Fisher Scientific]; and 500 μg/mL proteinase K [QIAGEN]), followed by complete homogenization with a TissueLyser LT (QIAGEN) using a ball bearing. After a 30-min incubation at 55°C, standard QIAzol (QIAGEN) protocol for tissue extraction was performed along with RNA quantification with a NanoDrop 2000c (Thermo Scientific), and RNA integrity was verified on a 2100 Bioanalyzer (Agilent Technologies). cDNA was then produced using Quantitect Reverse Transcription (QIAGEN) on 200 ng of RNA, and RT-qPCR was used to determine mRNA levels for DMPK and Dmpk mRNA, with Hprt1, Rpl13a, and Tbp RNAs as normalization controls (primers in Table S3 ) on a LightCycler 480 II (Roche) using SYBR Green I Master hot start reaction mix.
Tris cl
Manufactured by Wisent
Tris-Cl is a buffer solution commonly used in molecular biology and biochemistry applications. It is a mixture of tris(hydroxymethyl)aminomethane (TRIS) and hydrochloric acid (HCl) that creates a pH-stable environment for various laboratory procedures.
Automatically generated - may contain errors
Lab products found in correlation
Sodium dodecyl sulfate (sds), by Roche (2 mentions)
Proteinase k, by Qiagen (2 mentions)
Sodium chloride (nacl), by Thermo Fisher Scientific (2 mentions)
Mgcl2, by Thermo Fisher Scientific (2 mentions)
Nanodrop 2000c, by Thermo Fisher Scientific (2 mentions)
Ethylene diamine tetraacetic acid (edta), by Merck Group (2 mentions)
Proteinase k, by Wisent (1 mentions)
2100 bioanalyzer, by Agilent Technologies (1 mentions)
Lightcycler 480 2, by Roche (1 mentions)
Tissuelyser lt, by Qiagen (1 mentions)
Qiazol, by Qiagen (1 mentions)
Quantitect reverse transcription, by Qiagen (1 mentions)
Dab chromogen, by Abcam (1 mentions)
2 protocols using tris cl
1
Comprehensive RNA Isolation and Analysis
2
Immunochemical Analysis of ASO Distribution in Mouse Brain
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The distribution of ASO in mouse brain tissues was assessed by immunochemistry as described previously [66 (link)]. Briefly, fixed brain sections from 3 mice per group were digested for 1 min at 37 °C with a solution containing 500 mg/mL of proteinase K (Qiagen), 10 mM Tris-Cl, pH 7.5 (Wisent Bioproducts), 10 mM EDTA (Millipore Sigma), 2% SDS (Roche Diagnostics), 500 mM NaCl (Thermo Fisher Scientific), and 1.5 mM MgCl2 (Thermo Fisher Scientific). They were then blocked with 3% BSA for 30 min. After two washes in phosphate-buffered saline, the samples were incubated with a polyclonal rabbit anti-ASO primary antibody (6651 Pan ASO; Ionis Pharmaceuticals Inc., Carlsbad, CA, USA) for 1 h at room temperature and then with a goat anti-rabbit IgG (HRP) secondary antibody. The DAB chromogen (Abcam, Toronto, ON, Canada) was applied for 5 min before nuclear counterstaining with hematoxylin.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!