Promega eNotes - Applications

These experimental procedures cover four major steps from RNA isolation through T-vector cloning of the PCR amplification product. RNA was isolated from mouse and rat brain using the SV Total RNA Isolation System^(a) (Cat.# Z3100) and the PolyATtract^Ž System 1000(Cat.# Z5420). Denaturing agarose gel electrophoresis and Northern blot analysis of isolated RNA was performed. RT-PCR analysis was performed on total RNA from mouse and rat brain, as well as rat poly(A)+ RNA. T-vector cloning of beta-actin RT-PCR product was performed followed by colony screening for positive recombinants. These experiments were developed as part of the test experiments for use in the Molecular Approaches to Neuroscience course, offered by the BioPharmaceutical Technology Center Institute and Promega Neurosciences.

The isolation and characterization of nucleic acids entails basic procedures in the molecular biology repertoire. In neurobiology, though, the overwhelming tissue source is brain, which can present special problems such as the presence of high amounts of lipids and the fact that many messages of CNS origin are rare. The ability to easily and effectively obtain nucleic acids---DNA and RNA---from brain tissue is critically important in many neurobiology experiments. In this brief report, it is demonstrated that the isolation of total and messenger RNA (mRNA) from brain tissue can be accomplished with straightforward procedures and reagents, which are available commercially.

Total RNA was isolated from mouse and rat brain using the SV Total RNA Isolation System. Poly(A)+ RNA was isolated using the PolyATtract^Ž System 1000. For total RNA isolation, 175ľl of SV RNA Lysis Buffer per 60mg of brain were added to a 50ml conical tube. Brain tissue was placed into the tube and homogenized to completely disrupt the tissue. RNA was isolated from 175ľl of the generated lysate following the provided spin format SV Total RNA Isolation System protocol (#TM048). Poly (A)+ RNA was isolated using Promega’s PolyATtract^Ž System 1000 following the recommended protocol (#TM228). Typical yields and purity are listed in Table 1.

Mouse and rat total RNA was analyzed by denaturing agarose gel electrophoresis. In this procedure, 2.5ľg of RNA was denatured and resolved in an agarose gel containing formaldehyde. After electrophoresis, the resolved RNA samples were visualized by ethidium bromide staining. As evident in Figure 1, Panel A, bands representing ribosomal RNA (rRNA) subunits (18s and 28s) present in the total RNA samples were clearly visible. If the total RNA sample is undegraded, the 28s rRNA subunit band will appear approximately twice as intense as the 18s rRNA subunit band.

Figure 1. Analysis of total RNA from mouse and rat brains. Panel A: Total RNA was resolved by denaturing agarose gel electrophoresis. Lane 1, mouse total RNA (2.5ľg); lane 2, rat total RNA (2.5ľg); lane M, RNA Markers, 0.28-6.58kb (Cat.# G3191). Panel B: Northern blot of gel depicted in Panel A blotted and probed for G3PDH mRNA. (Marker lane not shown.)

The RNA was transferred to a membrane by upward capillary transfer and the resulting Northern blot was analyzed for specific message by detection with an RNA probe (Riboprobe^Ž System-T7^(b), Cat.# P1440) for mouse glyceraldehyde-3-phosphate dehydrogenase (G3PDH) mRNA. A specific band of the appropriate size (~1.2kb) was detected, as shown in Figure 1, Panel B.

RNA was next analyzed by reverse transcriptase-PCR (RT-PCR) using Promega’s Access RT-PCR System^(c) (Cat.# A1250). One microgram of mouse total RNA, rat total RNA or 10ng of rat poly(A+) RNA were amplified using Beta-Actin (Cat.# G5740) or NGF (Cat.# G5780) Primer Pairs^(c) as indicated. (A separate beta-actin primer pair [B2] distinct from Cat.# G5740 was also used.) Expected product size was 189bp for the NGF Primer Pair, 285bp for the Beta-Actin Primer Pair, and 511bp for the B2 primer pair.

Reverse transcription was performed for 45 minutes at 48°C. The cDNA amplification program consisted of an initial denaturation step (94°C for 2 minutes) followed by 40 amplification cycles of denaturation (94°C for 30 seconds), annealing (60°C for 1 minute) and extension (68°C for 1 minute). A final extension at 68°C for 7 minutes was included. Equivalent volumes of each amplification reaction were resolved on a 1.5% agarose gel as evident in Figure 2. Negative control reactions that contained Promega’s NGF or Beta-Actin primers with no RNA resulted in no product (lanes 7 and 8).

Figure 2. RT-PCR analysis of mouse and rat total RNA and rat poly(A)+ RNA. Lanes 1 and 2, mouse total RNA; lanes 3 and 4, rat total RNA; lanes 5 and 6, rat poly(A)+ RNA, lanes 7 and 8, no RNA. Beta-Actin Primer Pair was used for reactions represented in lanes 2 and 8. NGF Primer Pair was used for reactions represented in lanes 1, 3, 5 and 7. Beta-actin [B2] primer pair was used for reactions represented in lanes 4 and 6 (511bp expected product). Lane M, Promega’s 100bp DNA Ladder (Cat.# G2101).

T-vector cloning was performed using the pGEM^Ž-T Easy VectorSystem^(d,e) (Cat.# A1380) and mouse RT-PCR product generated using the Beta-Actin Primer Pair (reaction represented in lane 2 of Figure 2). Ligation reactions of the PCR product and pGEM^Ž-T Easy Vector were transformed into JM109 competent bacteria and plated onto LB plates containing ampicillin (100ľg/ml), IPTG (0.5mM) and X-gal (80ľg/ml) for blue-white color screening. After overnight growth at 37°C, blue, pale blue and white colonies (presumed to contain beta-actin insert) were rapidly screened by PCR using Tfl DNA Polymerase^(c) (Cat.# M1941). Each colony was picked and placed into 30ľl of LB broth + ampicillin. Two microliters of each culture were then placed directly into PCR mixes that contained the beta-actin primer pairs and amplified for 30 cycles under the following conditions: initial denaturation at 94°C for 2 minutes, followed by 30 cycles of denaturation (94°C for 30 seconds), annealing (60°C for 1 minute) and extension (68°C for 1 minute). A final extension at 68°C for 7 minutes was included. Equivalent volumes of products were resolved on a 2% agarose gel and visualized by ethidium bromide staining.

These experiments demonstrate the integration of many core Promega products for the isolation and analysis of nucleic acids in neurobiology. The protocols used were as recommended in the Technical Bulletins or Manuals for each product. The reagent amounts for RT-PCR are listed below. These procedures have been used successfully in the Promega Neurosciences' training course, Molecular Approaches to Neuroscience. For more information on the neuroscience training course, see the eNotes Feature article, "Cooking in a Better Kitchen: Neuroscientists Go Molecular."

Component	Volume
Sample in water	32ľl
Downstream Primer	1ľl
Upstream Primer	1ľl
AMV/Tfl 5X Buffer	10ľl
dNTP Mix (10mM each)	1ľl
25mM MgSO₄	2ľl
Tfl DNA Polymerase^(c)	2ľl
*For RT-PCR, add AMV RT	1ľl
Total	50ľl

^(b)U.S. Pat. No. 5,552,302 and other patents. Inhibitors of Angiogenin, which comprises a segment of human PRI, is the subject of U.S. Pat. No. 4,966,964 and other patents assigned to the President and Fellows of Harvard College and exclusively licensed to Promega Corporation.

^(c)The PCR process is covered by patents issued and applicable in certain countries. Promega does not encourage or support the unauthorized or unlicensed use of the PCR process. Use of this product is recommended for persons that either have a license to perform PCR or are not required to obtain a license.

^(e)Licensed under one or both of U.S. Pat. No. 5,487,993 and European Pat. No. 0 550 693.

pGEM, PolyATtract and Riboprobe are trademarks of Promega Corporation and are registered with the U.S. Patent and Trademark Office.

Focus: RT-PCR

Measuring Gene Expression from Mammalian Brain Tissue

By Dan Kephart, Ph.D.