View source for Preparation of RNA (In Vitro Transcription) (Sive et al. 2000)

'''Information adapted from:'''

''Early Development of Xenopus Laevis: A Laboratory Manual.''[http://www.xenbase.org/literature/book.do?method=display&bookId=22]

2000; (First ed.). Cold Spring Harbor: Cold Spring Harbor Laboratory Press. 

Sive, Hazel (Author), Grainger, Robert M (Author), and Harland, Richard M (Author).


The methods presented here have been adapted for use on Xenopus embryos. The more generally applicable methods are not provided. Although many of the protocols listed call for treatment with DEPC (diethylpyrocarbonate), this id generally not necessary when starting with good quality sterile distilled water.


'''RNA Isolation'''


'''Preparation of RNA (In VItro Transcription)'''


RNAs produced by in vitro transcription can be introduced into embryos by microinjection and used to test the effects of overexpression, misexpression, and expression of dominant-negative constructs. This method (adapted from Green et al. 1983) assumes that the coding region of interest is already inserted behind and appropriate bacteriophage promoter. A cap analog, GpppG, is included in the nucleotide mis to result in capped RNA.


1. For a 20-ml reaction volume, assemble the following ingredients, in a prewarmed tube, in the order indicated. If the nucleotide precipitates before synthesis, dilute and warm the reaction.


:linear template DNA (0.5-1 mg/ml) - 2 ul
:2x mRNA nucleotide triphosphate mix (see [http://www.xenbase.org/xenwiki/index.php/DNA/RNA_preparation]) - 10 ul
:alpha-32P UTP (if desired) - 0.1 ul (trace)
:DTT (200 mM) - 2 ul
:BSA (1 mg/ml) - 2 ul
:10x mRNA transcription buffer (see [http://www.xenbase.org/xenwiki/index.php/DNA/RNA_preparation]) - 2 ul
:RNase inhibitor (20 units/ml) - 0.5 ul
:bacteriophage RNA polymerase (20 units/ml) - 1.5 ml


Note: A precipitate invariably forms during the reaction, but the yields and bioactivity are good.


2. Mix gently and incubate the reaction for 2 hours at 37 deg C.


3. Add 2 units of RNase-free DNase I and incubate for a further 10 minutes at 37 deg C. Cerenkov count (i.e., with no scintillation fluid) the tube to obtain the total available cpm.


4. Dilute the reaction to 100 ul with a buffer containing 100 mM NaCl, 30 mM EDTA, 20 mM Tris (pH 7.5) and 1% SDS.


5. Purify the RNA through a spin column (Qiagen or Sephadex G-50).


Note: The column removes unincorporated nucleotides and cap analog, which would otherwise aggregate at the interphase of a phenol/choloroform extraction and necessitate multiple extraction. The cap analog is extremely toxic to embryos, so must be removed efficiently. Removal of the unincorporated nucleotides allows the efficiency of the reaction to be measured with the elute. If the reaction has not worked well, the counts will remain in the column.


6. Extract the column eluate with phenol:chloroform and check the recovery counts from the interface and, if necessary, extract again. Precipitate the aqueous phase with 2.5 volumes of ethanol and 0.1 volume of 3 M sodium acetate (pH 5.5).


7. Collect the precipitate by centrifuging for 15 minutes at maximum speed in a microcentrifuge at 4 deg C. Redissolve the pellet in 100 ul of DEPC-treated water and take a 1-ul aliquot for scintillation counting. Calculate the total incorporated cpm to obtain the percentage incorporation.


8. (optional) Reprecipitate the RNA in 2.5 volumes of ethanol and 0.1 volume of 3 M sodium acetate (pH 5.5). Wash the pellet in 70% ethanol. Resuspend at desired concentration.


9 Load 1 ul of the transcription mix onto a denaturing agarose gel (see note below) to assess the transcript yield and its integrity. Check the final resuspension of RNA by recovery counts, if radioactivity has been used.


Note: A simple denaturing agarose gel uses a standard Tris-acetate buffer but denatures the sample prior to loading.


The sample should be heated at 65 deg C for 10 minutes in:
:50% formamide
:2.2 M (6.7%) formaldehyde from a 37% stock
:1x MOPS buffer
:0.05% bromophenol blue
:100 ug/ml ethidium bromide

This is conveniently done using a stock of "FFM"


FFM
:10 ml formamide
:3.5 ml formaldehyde
:2.5ml MOPS buffer
:bromophenol blue/xylene cyanol (0.05% each)
:ethidium bromide (1 mg/ml)


10x MOPS
:0.2 M MOPS (ph7.0)
:50 mM sodium acetate
:5 mM EDTA


After electrophoresis, the gel can be photographed directly.


Helpful Hints:


*Problems with Yield and Translation


Poor yield is usually due to poor template, although occasionally commercial reagents can be at fault. Some batches of GpppG inhibit transcription, or worse, they promote synthesis of untranslatable RNA. It can be useful to keep stocks of known good quality reagents as positive controls, which will provide a basis for complaints to the manufacturer.


* Calculation of Yield


If the labeled nucleotide is UTP, then the reaction above contains 10 ul of 6 mM UTP. In an average RNA, for every mole of UTP incorporated, there will be one mole of each of the other three nucleotides incorporated. The average molecular weight of nucleotide monophosphate is assumed to be 330 g/mole. If all the UTP were incorporated, the tiled would be: (10 x 10^-6 liters) x (6 x 10^-3 moles/liter) x 4 x 330 g/mole = 79.2 ug, i.e., (volume of label) x (concentration of label) x (number of nucleotides) x (average molecular weight of each nucleotide).


For the purposes of calculating mass, each 1% yield corresponds to 0.8 ug of RNA. For synthesis if large numbers of RNAs, e.g., as in expression cloning, half-scale transcription reactions can be performed. For a half-scale reaction, each 1% incorporation of UTP corresponds to 0.4 ug of RNA.


mRNAs of higher specific activity can be synthesized without excessive radiolytic degradation. Radioactive nucleotide (10 uCi) can be added to the standard reaction. The RNA will retain approximately 25% of its original activity after storage for 2 years at -80 deg C in DEPC-treated water.


*Choice of Vector


All polymerases add dinucleotide cap with the same efficiency (based on stability of RNA in oocytes). However, some polylinkers contain sequences that appear to inhibit translation (Kuo et al. 1996). The effect is observed when cDNAs containing 5' dC trances are cloned downstream from T7 or T3 polymerase promoters. The effect is thought to be due to formation of secondary structure between the initial GGG triplet of T3 and T7 transcripts, and the oligo(dC) tracts. Since SP6 transcripts initiate with AAA, and are not capable of forming this secondary structure, their translation is unaffected. In general, the less polylinker present, the better.


DNAs for translation are often cloned into plasmids that contain globin 5'UTR (untranslated region) and 3'UTR, e.g., pSP64 or derivatives. Although no systematic analysis has been published, in some cases, they appear to have no effect. These plasmids may be partially useful for the overexpression of maternal mRNAs. Regulation of maternal RNA expression is mainly translational, and regulatory regions are often contained in the 3'UTR and 5'UTR stretches. Removing these regions and substituting the strong globin UTRs, or even the AUG Kozak consensus sequence, may help significantly.


The CS series of vectors (Turner and Weintraub 1994) incorporate a cytoplasmic polyadenylation signal in the primary transcript. Cytoplasmic polyadenylation (Richter 1991) enhances translation tenfold, or more, compared to that achieved by equivalent transcripts from pSP64T. Conversely, for oocyte injections, where translation occurs during long-term incubation, pSP64T transcripts, which are polyadenylated in vitro, are translated more efficiently than the equivalent CS transcripts polyadenylated in vivo (after injection into the oocyte nucleus). In vitro polyadenylation by poly(A) tracts encoded by the vector provides a useful means by which to select full-length transcripts, using oligo (dT) cellulose chromatography if desired. However, poly(dA) tracts encoded into the 3' poly linker of a plasmid can be unstable in plasmids, more than 50 As are seldom maintained.