Total RNA

The only method used to quantitate ribonucleic acid is the orcinol assay.20 With this method, RNA is depurinated in concentrated HC1 and the resulting ribosephos-phates are dephosphorylated and dehydrated to produce furfural (Eq. 1.5). Furfural then reacts with orcinol in the presence of Fe3+ to yield colored condensation products, as shown in Eq. 1.4, which together possess an absorption maximum at 660 nm. 

The DNA also undergoes a limited reaction under these conditions, and yields 10% of the color of a similar concentration of RNA the dehydration step to form furfural is not readily accomplished by 2-deoxyribose. 

The original orcinol assay has been modified to improve its selectivity toward RNA over DNA and sugars. The improved method21 uses a 1.0-mL RNA sample incubated 24 h at 40 °C with 4.0 mL of 85% sulfuric acid prior to addition of the orcinol reagent (containing no Fe3+). Under these conditions, colorimetric measurements are made at 500 nm, where products formed by the reaction of levulinic acid (from DNA) with orcinol do not absorb. Proteins (e.g., BSA) do not interfere, and the method is sixfold more selective to RNA over DNA than the original orcinol assay. 

---

A relatively large amount of RNA needs to be labeled with fluorescent dyes in order to obtain satisfactoiy results (20-100 pg of total RNA or 1-2 pg of mRNA). These requirements could make microarrays incompatible with the very limited RNA yields obtained from some... 

Amplification based methods can be used with as little as nanograms of total RNA (corresponding to <1000 cells) whereas the dendromer-based methods need at least one microgram of total RNA. 

B. Northern blot analysis of total RNA extracted from hypocotyls of P. vulgaris and hybridised to a radioactive pg/p-specific probe. 

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.

A.aculeatus CBS 115.80 was grown on apple pectin in a fermenter and strong induction of Rgase production could be observed. Total RNA was isolated from such an RGase producing culture and used for the construction of a cDNA library. 

Total RNA was isolated from mycelia cultured 6 days on PG-inducing and non- inducing conditions (apple pectin and glucose, respectively). The RNA extraction was performed according to the phcnol-SDS method combined with selective precipitation using LiCl. RNA concentration was estimated by absortion at 260nm. 

Fig. 19.1 Differential displays comparing RNAs from saline (S)-, imipramine (I)- or fluoxetine (F)-treated rats. Total RNA was extracted from hypothalami of animals treated with the different drugs for two months. Autoradiograms of amplified -[35S]-dATP-labeled PCR (polymerase chain reaction) products after electrophoresis in 6% polyacrylamide gels are shown for two different primer combinations that identified one upregulated (arrowhead) and one downregulated (arrow) fragment in the groups treated with antidepressants (from [4] with permission).

Incubate >1 /ig of RNA with a 3- to 10-fold molar excess of radio-labeled probe (about 150 to 600 pg per 10 /ig total RNA) in hybridization buffer. 

An example experiment is shown in Fig. 6.3A. Here, a dual probe RPA was performed on 1 /ig of total RNA purified from cells transfected with cap tail R-luc-4 sites and F-luc mRNA, with and without miCXCR4. The assay was performed on total RNA from untransfected cells to test for specificity. The level of R-luc-4 sites mRNA was not significantly different between cells transfected with miCXCR4 (+) and those not (—). 

Comments There are several suggested controls for this assay, including use of yeast total RNA as a negative control (check for probe species specificity) and a no RNAse control to determine probe stability. In Fig. 6.3A, the positive control marker lane was produced by addition of R-luc-4 sites or F-luc mRNA only to the assay. Also, optimal times for RNAse digestion will vary from probe to probe. In addition, for maximum sensitivity a probe with high specific activity is preferable (yet still in molar excess to the mRNA). 

DNasel treat total RNA using the Turbo DNA-free reagent (Ambion). 

To quantify the amount of firefly and Renilla luciferase RNAs, total RNA is extracted from transfected neurons, reverse transcribed, and subjected to real-time PCR amplification using QuantiTech SYBR Green PCR mixture (Qiagen). 

Fig. 3.3 A. Northern blot showing the synthesis of Rubisco SSU mRNA after 24-36 h of sprouting in an airlift tank. Total RNA was isolated from sprouts germinated from 12 to 168 hours. B. On the same filter, unlabelled Rubisco RNA produced by in vitro transcription was loaded as a control. The amount of control RNA is indicated in pg.

Following a procedure modified from the protocol of Newman [Newman et al. 1990], total RNA was extracted from 100 ml C. moewusii culture after 2 hours of anaerobic adaptation. The mRNA was isolated from 250 pg of total RNA using the Oligotex mRNA Mini Kit (Qiagen, Hilden, Germany). 50 pg RNA isolated from anaerobically induced cultures of C. moewusii (1 h, 2 h, 3 h and 4 h) were separated on a 0.8 % agarose gel... 

Using 0.2 kb of the 3 UTR region of hydAl, a RNA probe was generated and hybridised with total RNA which had been derived from 50 ml culture samples after 0, 1, 2 and 4 hours of anaerobic induction. The Northern blot results demonstrate that hydAl is highly transcribed under conditions of anaerobic adaptation (Fig. IB). 

A Anaerobic adaptation was obtained by flushing the cells with argon. At indicated time points, samples were taken to measure the in vitro Fk-ase activity of C. reinhardtii (box), S. obliquus (circle), C. fusca (triangle), C. moewusii (diamond). While the activities of both Scenedesmus species are comparative low, the in vitro H -production rate of anaerobically induced C. moewusii cultures is 2 times higher than the activity of induced C. reinhardtii cultures. B Northern blots with equal amounts of total RNA isolated from an anaerobically adapted culture (2 h) and an uninduced reference culture (0 h) of C. moewusii. The upper blot was incubated with a RNA sample of the 3 UTR of the hydA 1 cDNA, while the lower blot was incubated with a RNA sample generated of the cDNA from the constitutively expressed sedoheptulose 1,7-bisphosphatasegene. 

Adapted cells were harvested at 0 h, 1 h, and 3 h, and total RNA was isolated as described in Materials and Methods. Northern hybridization with the ars specific probe (A) and the hydA specific probe (B) are shown. 

B) RNA gel blot hybridization revealed massive enhancement of the PAP1 expression. Total RNA was isolated from 4-week-old papl-D and wildtype plants. 

Figure 7.10. IL-l/J expression by GM-CSF-stimulated neutrophils. Human neutrophils were incubated in the absence (-) and presence (+) of 50 U/ml GM-CSF, and at time intervals total RNA was extracted. After electrophoresis on formamide-containing gels, IL-1/3 mRNA levels were probed. Source Experiment of Julie Quayle.

CNTs can conjugate with nucleic acids via non-covalent bond. ssDNA, short double-stranded DNA and total RNA molecules can attach to the surface of CNTs and can disperse CNTs in aqueous environment. The poly(30T) has the highest dispersion efficiency (Zheng et al., 2003). For example, 1 mg DNA molecules mix with lmg CNTs in 1ml water, yield at most 4mg/ml CNT solution. DNA-CNT complexes can be purified or isolated by electronic properties such as agarose gel electrophoresis and centrifuge method (Cui et al., 2004a Karajanagi et al., 2004). 

Total RNA can be prepared using commercially available kits according to the protocols of the manufacturer or by the guanidinium isothiocyanate method, followed by caesium chloride centrifugation (CsCl). Details are published in handbooks of molecular biology techniques. In our hands, both methods are equivalent, but if budgets are limited, the CsCl method may be more suitable for the preparation of large amounts of RNA. 

During recent years, many of our appheations of competitive RT-PCR have involved the analysis of RNA from very small tissue samples obtained by laser capture microdissection. We have therefore adopted a modified reamphfication protocol (competitive Nested-RT-PCR) for the quantitation of very small tissue samples (Costa et al., 2002). The procedure involves the use of very low concentrations of cRNA (internal standard) in the presence of total RNA isolated from a measured and microdissected tissue sample or a... 

---