Journal: Nucleic Acids Research
Article Title: RNA structure analysis assisted by capillary electrophoresis
doi:
Figure Lengend Snippet: Analysis of structurally heterogeneous BRCA1 Ex1a and transcript truncation to obtain a homogeneous structure. ( A ) Non-denaturing 10% polyacrylamide gel electrophoresis of 5′-end radiolabeled Ex1a transcript treated as follows: lane 1, dissolved in water and incubated at 20°C for 30 min; lane 2, heated to 75°C for 1 min (denaturation) and cooled slowly to 20°C (renaturation); lane 3, dissolved in the structure-probing buffer (10 mM Tris–HCl pH 7.2, 10 mM magnesium ions, 40 mM NaCl) and incubated at 20°C for 30 min; lane 4, dissolved as described for lane 3 and subjected to the denaturation/renaturation procedure; lane 5, dissolved as described for lane 3, carrier RNA added to a final concentration of 8 µM, and incubated at 20°C for 30 min; lane 6, carrier RNA added and subjected to denaturation/renaturation. ( B ) CE in non-denaturing conditions of Ex1a transcript fluorescently labeled at its 3′ end with TdT and: [R110]dUTP, [RG6]dUTP, [TAMRA]dUTP (shadowed peaks); TAMRA-500 internal standard (gray line). ( C ) CE in non-denaturing polymer at temperatures: 30, 45 and 60°C of Ex1a transcript end labeled with [R110]dUTP and Klenow fragment. ( D ) Non-denaturing 10% polyacrylamide gel electrophoresis of 5′-end radiolabeled Ex1a transcript (0.5 µM) (lane 1), and the same transcript hybridized with 18 nt of Rex1a oligodeoxynucleotide complementary to its 3′ end (lane 2). Hybridization of transcript (1 µM) with oligodeoxynucleotide (1 µM) was performed in 15 mM Tris–HCl (pH 7.2), 10 mM MgCl 2 , 1.5 mM DTT by heating the sample at 90°C for 1 min and fast cooling. Arrowhead indicates the position of hybrid migration. ( E ) CE in non-denaturing conditions of Ex1a102nt transcript labeled with TdT and [RG6]dUTP (gray line indicates ROX-500 internal standard).
Article Snippet: The standard labeling reaction with Klenow fragment was performed in a 10–20 µl volume and contained: 5 µM gel-purified transcript, 20 µM oligodeoxynucleotide templates (ex1a47nt-fl 5′- CA GTCCAGGAAGTCTCA, ex1b64nt-fl 5′- TA GGACACTCAGTGCCC, ex1a-fl 5′- CA CTTTACCCAGAGC and ex2-fl 5′- CA CAGATGGGACAC) complementary to the 3′ end of the four RNA molecules to be labeled: Ex1a47nt, Ex1b64nt, Ex1a and Ex1a-2, respectively (non-complementary positions underlined), 20 µM labeled dUTP (PE Applied Biosystems), 50 mM Tris–HCl (pH 7.2), 10 mM MgSO4 , 0.1 mM DTT, 20 U RNazin and 10 U Klenow fragment (Promega).
Techniques: Polyacrylamide Gel Electrophoresis, Incubation, Concentration Assay, Labeling, Hybridization, Migration