Northern blotting, nuclease protection assays and RT-PCR

In relative quantitation, RNA levels of the gene of interest are compared from sample to sample using an internal control to normalize for differences in sample concentration and loading. As the internal control should be expressed at a constant level across all samples, both choosing and authenticating this control are critical to successful relative quantitation. In the paragraphs below, we describe commonly used internal controls and how these are authenticated.

Internal standard RNA probes
Inconsistencies in RNA isolation and in the commonly used RNA analysis procedures listed above can introduce errors into the analysis process. One method for minimizing these errors is to simultaneously measure a cellular RNA that has a constant expression level between samples. This RNA serves as an internal standard or reference against which other RNA values can be normalized. This process corrects for sample to sample variation.

The ideal RNA species for an "internal standard" should be expressed at a constant level across all of the types of samples being analyzed. For example, the internal standard should be expressed equally among different tissues of an organism, at all stages of development, and for both control and experimentally treated cell types. A constitutively expressed "housekeeping" gene would appear to be a good model for an internal standard RNA. Unfortunately, there is no one single RNA with a constant expression level in all of these situations (although 18S rRNA appears to come close to being an ideal internal control under the broadest range of experimental conditions, see below). It is therefore necessary to identify the appropriate control RNA for the particular set of experimental RNA samples to be studied.

ß-actin
ß-actin mRNA was one of the first RNAs used as an internal standard. It encodes a ubiquitous cytoskeleton protein and is expressed at moderately abundant levels. In a 10 µg sample of total RNA, there is approximately 300 pg of ß-actin mRNA, representing 0.1% of mRNA or 0.003% of total RNA. This is compared to 0.3–3 pg of a rare mRNA species. The ß-actin gene is highly conserved in eukaryotes (1) and expressed in most cell types (2). However, the level of expression has been shown to vary in some tissues, including cultured adipocytes (3), mammary epithelial cell lines, breast fibroblast cell lines (4), and cultured human colon carcinoma cells (5). Our scientists have shown ß-actin mRNA levels to be high in normal mouse spleen, brain, embryo, heart, hypothalamus and kidney and relatively lower in mouse liver, lung and muscle (Figure 1).

Figure 1. Expression level of several common internal standards compared across mouse tissues using a ribonuclease protection assay (RPA). High specific activity antisense RNA probes (1.15 x 10⁹ cpm/µg) to GAPDH, ß-actin and cyclophilin were synthesized from Ambion^TM pTRI-GAPDH, pTRI-beta-actin, and pTRI-cyclophilin mouse DNA templates, respectively, in a MAXIscript in vitro transcription reaction. 4 x 10⁴ cpm of each probe was added to 5 µg total RNA from different mouse tissues and processed in a multiple probe ribonuclease protection assay using the RPA II Kit. Results show a comparison of the relative expression levels of each probe within a given tissue and across mouse tissues. Control lanes of yeast RNA and probe +/- RNase are shown relative to the ladder of molecular weight standards on the right.

GAPDH
Glyceraldehyde-3-phosphate-dehydrogenase (GAPDH) is a key enzyme in glycolysis. The mRNA encoding GAPDH is another moderately abundant message in cells, with expression levels similar to that of ß-actin. This housekeeping gene is constitutively expressed in many tissues and has been reported to be a useful internal control. GAPDH is expressed at high levels in rat and human muscle and heart (6) and has been used frequently as a control in experiments using cell lines (7, 8, 9). However, GAPDH levels vary with developmental stage and with dexamethasone treatment in embryonic chick heart and tendon cells (10). Furthermore, GAPDH mRNA levels varied significantly in several different virally-transformed or oncogene-transfected mouse fibroblast cell lines (11), during the cell cycle of normal human skin fibroblasts (12), in rat vascular smooth muscle cells treated with vasoconstrictor or mitogens (13) and some breast cell lines (4). GAPDH RNA levels have also been shown to increase significantly in cultured adipocytes treated with insulin (14). Our research has shown GAPDH to be expressed at relatively low levels in normal mouse liver, spleen, lung, embryo and hypothalamus and at relatively high levels in mouse brain, muscle, heart and kidney (Figure 1).

Cyclophilin
A third constitutively expressed gene, cyclophilin, encodes a ubiquitous cytoplasmic protein that plays a role in protein folding through the isomerization of peptide bonds. It is also known to bind cyclosporin-A and may mediate the drug's immunosuppressive effects (15). Cyclophilin mRNA is also somewhat abundant, comprising close to 0.1% of cytoplasmic mRNA, and is highly conserved across different species. It is expressed at high levels in normal rat brain, spleen, thymus, adrenal glands, ovaries and testes; however, lower level expression is seen in rat liver, lung and anterior pituitary (16). Cyclophilin is also expressed at relatively high levels in many monkey tissues except skeletal muscle (16). Some tumor cell lines have shown a higher concentration of cyclophilin mRNA than normal cells (15). However, no alteration of cyclophilin levels have been observed when T-lymphocytes are treated with mitogens and tumor promoters (15). Our researchers have seen cyclophilin mRNA expressed at similar high levels in normal mouse brain, thymus, ovary, kidney and embryo and relatively lower levels in mouse liver, heart, lung, spleen and testes (Figure 1). When compared with ß-actin and GAPDH, cyclophilin mRNA is less abundant across all mouse tissues tested (Figure 1). Given this fact, cyclophilin may prove to be a more appropriate internal control in multiprobe assays when studying the expression of rare mRNA species.

rRNAs
18S and 28S ribosomal RNAs may also be used as internal controls. These RNAs are less likely to fluctuate under conditions that affect the expression of mRNAs. This is because they make up 80% of a total RNA sample, such that when the concentration of a total RNA sample is determined from spectrophotometric readings, the sample is essentially already being normalized to the amount of rRNA it contains. rRNAs are also transcribed by a distinct polymerase from mRNAs, which may result in a different pattern of regulation of expression. Specifically, the expression levels of 28S rRNA have been observed to remain stable while levels of ß-actin and/or GAPDH vary. This has been seen in individual rat livers (17), normal human skin fibroblasts (12), malignant mouse cell lines (11) and some breast cell lines (4). Our scientists have seen 18S levels to be uniform in all mouse tissues tested. These include liver, brain, thymus, heart, lung, spleen, testes, ovary, kidney and embryo (Figure 2). In a 10 µg total RNA sample, 2 µg are contributed by 18S rRNA, and 5.5 µg are contributed by 28S rRNA. Thus, if the researcher is defining an equivalent amount of RNA from two samples to be a constant mass amount (10 µg), then by definition, the ribosomal RNAs will be constant from 10 µg sample to 10 µg sample. (Note that in some situations it is more relevant to normalize RNA to a constant cell number or tissue mass.) The very high abundance of the rRNAs requires the researcher to generate a large amount of low specific activity antisense probe for detecting these RNAs. (Note that the exact amount of probe needed will depend on the size of the probe:target region of hybridization; 0.5–1 µg of probe containing a 100 nt rRNA sequence is needed to be in 5 molar excess of rRNA target when performing a nuclease protection assay on a 10 µg total RNA sample.) A few micrograms of low specific activity probe can be synthesized using the MAXIscript™ In Vitro Transcription Kits, but for large numbers of samples, this is most conveniently done using the MEGAshortscript™ Large Scale In Vitro Transcription Kit.

Of course, all of these standards described here have not been tested in every possible system. Therefore, it may be prudent to test more that one internal control to ascertain which is most appropriate for a given experimental system (see "Validating Your Internal Control", below).

Figure 2. Expression level of 18S rRNA compared across mouse tissues using a ribonuclease protection assay (RPA). A low specific activity antisense 18S ribosomal RNA probe was synthesized from the Ambion^TM pT7-18S DNA template in a MAXIscript™ in vitro transcription reaction. 3 x 10⁴ cpm of probe was added to 3 µg total RNA from different mouse tissue processed in a ribonuclease protection assay using the RPA II™ Kit. Results show the relative expression level of 18S rRNA across mouse tissues. The doublet represents partial renaturation of protected fragment due to high concentration of rRNAs. Signal intensity is best obtained by measuring both bands (see the Ambion^TM T7-18S Antisense DNA Specification Sheet for further explanation). Control lanes of yeast RNA and probe +/- RNase are shown on the right relative to the ladder of molecular weight standards (only the 100 nt band is seen) on the left.

Validating your internal control
Determine the validity of an internal control for your experimental system by doing the following pilot experiment. Replicates of each sample type (e.g., treated vs. untreated, different tissue or cell types) will be used to analyze the expression of the potential internal control.

Using 3 to 4 replicates will provide statistically significant results. Add the same amount of internal control probe or primers to each sample. Do the experiment, and evaluate the results. Differences seen between samples should not be greater than differences seen within replicates of each sample. Figure 3 shows a Northern experiment used to validate ß-actin as an internal control for samples from various tissues. The data suggest that ß-actin could be used as an internal control for comparing samples from embryo and thymus, or heart and liver, but that it is clearly not an appropriate internal control for comparing samples from embryo and heart, for example.

Figure 3. Authenticating an internal control. Total RNA (2.5 µg) from the indicated tissues was run on a denaturing agarose gel in quadruplicate. Panel A shows the gel stained with Ethidium bromide. The gel was then Northern blotted and hybridized with a radiolabeled probe for ß-actin. The autoradiogram is shown in Panel B.

After hybridizing with the internal control, the samples are standardized and mRNA quantitation is expressed in relative terms. For example, in Figure 4 the liver lane has a 0.5:1 ratio of S15: internal control signal, whereas in the spleen lane, the ratio is 1.5:1. This indicates that S15 is expressed at a 3X higher level in spleen.

Figure 4. Relative quantitation in a northern. Five µg of various mouse total RNAs were electrophoresed and transferred to a membrane. The membrane was then hybridized with two RNA probes - one complementary to the internal control, GAPDH, and the other to ribosomal protein S15. The blot was washed and exposed to film.

Relative quantitation using nuclease protection assays (NPAs)
The ribonuclease protection assay (RPA) is particularly useful for the simultaneous quantitation of multiple mRNA species in one sample. Using the Ambion^TM RPA III™ Kit, up to 12 different mRNAs can be detected and quantified in a single sample. Probes used for multiprobe analysis must be of significantly different size to allow resolution on a gel and cannot contain regions of intermolecular complementarity. Prior to analysis, each probe must be tested to ensure that after digestion, it produces a single band. One of the probes must be an invariant internal control to which the samples will be standardized.

High specific activity probes should be used for rare targets, and low specific activity probes are needed for moderately and highly expressed targets such as GAPDH, ß-actin and 18S rRNA. Altering the specific activity of probes reduces their signal intensity so that rare and abundant transcripts can be detected with a single exposure.

Figure 5 shows the varied expression pattern of 4 oncogene mRNAs across different tissue types. In this case, cyclophilin is used as an internal control to normalize signal within each of the samples.

Figure 5. Ribonuclease protection assay using mouse tissues stored in RNAlater™ stabilization solution. Various mouse tissues were stored in RNAlater stabilization solution for 1 or 4 weeks at 4°C. RNA was isolated from each tissue and analyzed using the RPA III™ Kit. Ten µg of total RNA was hybridized with 5 x 10⁴ cpm of each of 5 combined antisense RNA probes, digested with RNase and precipitated. Products were assessed on a 5% polyacrylamide/8 M urea gel and exposed to film for 4 hours at -80°C with an intensifying screen.

Relative Quantitation Using RT-PCR
Relative RT-PCR uses primers for an internal control that are multiplexed in the same RT-PCR reaction with gene specific primers. Internal control and gene specific primers must be compatible; that is, they must not produce additional bands or hybridize to each other. Additionally, for relative RT-PCR data to be meaningful, the PCR reaction must be terminated when the products from both the internal control and the gene of interest are being amplified within the linear range.

Our scientists recommends using 18S rRNA as an internal control; however, because of its abundance, rare message products could not previously be detected in the linear range of 18S rRNA. The Ambion^TM QuantumRNA™ 18S Internal Standards solve this problem. By mixing primers for 18S rRNA with our exclusive "competimers" — primers of the same sequence but that cannot be extended — the 18S rRNA signal can be reduced even to the level of rare messages.

Thermo Fisher Scientific has over 90 gene specific relative RT-PCR kits available that contain primer pairs for specific human, mouse, and rat genes and QuantumRNA 18S rRNA internal control primers. Typical data generated using these kits is shown in Figure 6.

Figure 6. Multiplex quantitative RT-PCR with Invitrogen^TM Ambion^TM gene specific relative RT-PCR kits. 1X (A) or 10X (B) amounts of RNA were reverse transcribed with the RETROscript™ Kit and random decamers. Individual PCR reactions were performed with the indicated Gene Specific Relative RT-PCR Kit, each reaction containing gene-specific primers, 18S rRNA primers and 18S rRNA Competimers™ primers. Competimer technology attenuates 18S rRNA amplification efficiency so that it can be multiplexed effectively with the much less abundant interleukin targets.

Absolute quantitation measures the absolute amount (e.g. 5.3 x 10⁵ copies) of a specific sequence in a sample. Dilutions of a synthetic sense strand RNA (identical or very similar to the target template) are co-amplified or detected along with the endogenous target. The sense RNA creates a concentration curve to which the endogenously expressed message is then compared in order to obtain an absolute measurement of the transcript under study.

Absolute quantitation using northern analysis
Absolute quantitation by northern analysis is straightforward. A sense strand RNA transcript complementary to the probe is synthesized (see the Ambion^TM MAXIscript™ in Vitro Transcription Kit). The synthetic RNA is quantitated, and dilutions are spiked into a yeast RNA background and size fractionated in a denaturing agarose gel next to the sample RNAs. Alternatively, a longer or shorter sense RNA, sharing target sequence with the endogenous message, can be added to the experimental samples. After blotting the membrane is probed with an antisense probe, and the amount of endogenous target is compared with the concentration curve generated by the synthetic sense strand dilutions.

Figure 7. Quantitation of variable ß-actin expression in mouse tissues. Mouse total RNA from several tissues was run on a northern gel and transferred to membrane using the Ambion™ NorthernMax™ Kit. Dilutions of full-length ß-actin sense RNA were spiked into a yeast background and also run on the same gel. A 32^P-labeled antisense ß-actin probe was used to detect ß-actin mRNA in the mouse tissues as well as the artificial sense strand. Absolute expression levels were determined by direct comparison of the signal from the mouse tissues with the concentration curve generated with artificial sense strand ß-actin.

Absolute quantitation using nuclease protection assays (NPA)
As with absolute Northern analysis, the quantitation curve for absolute NPA analysis is constructed by spiking quantitated sense strand RNA dilutions into a yeast RNA background. These control reactions are then processed along with the sample RNAs and the reaction products are separated on a denaturing polyacrylamide gel. Figure 8 shows an example of absolute quantitation using NPA analysis. In this experiment, approximately 10 pg of CAT mRNA are expressed per 100,000 pSVCAT transfected COS cells.

Figure 8. Detection of cloramphenicol acetyltransferase (CAT) expression in COS cells transiently transfected with pSVCAT. Increasing dilutions of pSVCAT-transfected COS cells were assayed for CAT by ribonuclease protection. Reaction products were resolved on a denaturing 8% polyacrylamide gel, dried, and autoradiographed. Lane 1, Ladder of molecular weight standards; lane 2, undigested CAT probe; lane 3, CAT probe digested after hybridization with total RNA from 4 x 10⁵ COS cells; lanes 4-9, CAT probe digested after hybridization with total RNA from 4 x 10⁵, 2 x 10⁵, 10⁵, 5 x 10⁴, 2.5 x 10⁴, and 1.25 x 10⁴ transfected COS cells; lanes 10-14, standard concentration curve generated by digestion of the CAT probe after hybridization to 10, 20, 50, 100, and 200 pg of truncated sense transcript diluted in 5 µg of yeast RNA.

Absolute or competitive RT-PCR
Competitive RT-PCR precisely quantitates a message by comparing signal intensity of the RT-PCR product to a concentration curve created using a synthetic transcript or "competitor". Competitors are designed to: 1. be amplified using the same primers as the desired target sequence; 2. have the same amplification efficiency as the target sequence; 3. be of a significantly different size from the target to allow differentiation of the two products on an agarose gel; and 4. control for variations in the RT reaction. Competitor RNA transcript is synthesized, quantitated and diluted into the sample RNA. Pilot experiments are run to determine the range of competitor concentration where the experimental signal is similar in concentration to the endogenous target. A more precise concentration can then be determined by repeating the experiment with a smaller dilution range, generating data that approximates abundance of the endogenous target in the sample. From the data illustrated in Figure 9, it can be determined that between 1.5 x 10⁶ – 3.05 x 10⁶ copies of hIL-10 RNA are present in the sample.

Figure 9. Competitive RT-PCR experiment. The indicated amounts of hIL-10 Armored RNA^TM competitor were added to 2 µg aliquots of experimental RNA, and the mixture underwent RT-PCR.

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