RNA blots

Figure 4. RNA Blot Analysis of pSubunit, PG, and D21 Expression during Fruit Development and Ripening. Total RNA (25 pg) isolated from the indicated tomato tissues was probed with eiAer a psubunit cDNA clone, a cDNA for the catalytic PG polypeptide, or a cDNA for the constitutively expressed mRNA D21. Identical specific activities were used in each hybridization and all blots were exposed for 8 hr.

Recommendations for blocking agents 5XDHS, all applications 10 X DHS, only for colony/plaque lifts BLOTTO, primarily for colony/plaque lifts yeast tRNA, primarily for RNA blots but not recommended for colony/plaque lifts homopolymer DNA, for AT- or GC-rich target DNA. 

Kong H, DePaoli AM, Breder CD, Yasuda K, Bell Gl, Reisine T (1994) Differential expression of messenger RNAs for somatostatin receptor subtypes SSTRl, SSTR2 and SSTR3 in adult rat brain analysis by RNA blotting and in situ hybridization histochemistry. Neuroscience 59 175-184... 

The term Southern blotting is used because the method was developed by E. M. Southern and the term northern blotting was introduced for RNA blots to distinguish them from DNA blots. More recently, protein chemists have gotten into the act by blotting electrophoresed proteins to a membrane and detecting specific protein molecules with antibodies, which is known as western blotting. Finally, southwestern blots (a renatured protein blot and a DNA probe) are used to study protein-DNA interactions. 

Chapters 17-22 describe the hybridization of the nonradioactive probes to the DNA and RNA immobilized on blots, together with the detection systems necessary to reveal where the probe has hybridized. Chapters 17-19 deal with digoxigenin probes, with Chapters 17 and 19 describing chemiluminescent detection on DNA and RNA blots respectively, and Chapter 18 describing a colorimetric detection system. Chapter 20 deals with enhanced chemiluminescent detection of enzymically labeled probes, whereas Chapters 21 and 22 describe enhanced chemiluminescent detection of large (Chapter 21) and small (oligonucleotide. Chapter 22) probes labeled with fluorescein. 

Serum stimulation of WI-38 cells. mRNA levels in the fibroblast cell line WI-38 induced to enter the cell cycle by serum addition to starved cultures were investigated. The amount of RNA blotted to the filter was monitored by hybridization to a control cDNA, which hybridizes to a 0.9 kb transcript, and has been shown to be expressed equally during aU phases of the cell cycle. Two major points emerge from the investigation of polymerase transcripts in this system. Even quiescent cells appear to contain basal levels of polymerase mRNA. Secondly, consistent with our earlier... 

RNA blot analysis. The la antigens, class II major histocompatibility antigens, are key molecules in sensitizing thymus-derived lymphocytes in the context of nominal antigens. Expression of la antigens on antigen... 

Fig. 3. RNA blot analyses with the cDNA for poly(ADP-ribose) synthetase and the 1 2 3 4 cDNA for la antigen. Total cellular RNA (20 pg per laneX isolated from mouse macrophage tumor P388D1 cells at the indicated culture period in the presence of 10 unit/ml interferon-y, was separated on a 0.8% agarose/formaldehyde gel and then blotted on to a nitrocellulose filter. The RNA on the filter was analyzed by the P-— labelled cDNA of poly(ADP-ribose) synthetase in panel A and the I-Ap cDNA...

In primary rat hepatocyte cultures 100 jiM ferric nitrilotriacetate induced five oxidation products of cellular DNA derived from both purines and pyrimidines (Abalea et al. 1999). Addition of increasing concentrations of myricetin (25-50-100 iM) simultaneously with iron prevented both lipid peroxidation and accumulation of oxidation products in DNA. Moreover, as an activation of DNA repair pathways, myricetin stimulated the release of DNA oxidation bases into culture media, especially of purine-derived oxidation products. The removal of highly mutagenic oxidation products from DNA of hepatocytes might correspond to an activation of DNA excision-repair enzymes by myricetin. This was verified by RNA blot analysis of DNA polymerase P gene expression, which was induced by myricetin in a dose-dependent manner. 

Figure 2. RNA blot analysis of jojoba elongase condensing enzyme gene expression in transgenic alfalfa. Total RNAs ( 15 gg) were separated on a formaldehyde gel, transferred onto a nylon membrane and hybridized with P-labeled jojoba condensing enzyme cDNA. RNA from vector transformed and nontransformed tissue culture-regenerated plants were used as control.

Measurement of carbon flux through pools of free CoA-SH and medium to long chain acyl-CoAs in leaf should provide valuable information about the potential differences in chloroplast and plastid fatty acid metabolism. Fatty acid breakdown in leaf is not required to any great extent until senescence, when the glyoxylate cycle uses P-oxidised fatty acid to produce 4-carbon acids which are converted to carbohydrates[5]. Currently, we are using enzyme assays. Western blotting and RNA blot analysis to determine if the glyoxylate pathway is functional in 35S-MCTE transformed Brassica. 

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