a total extract dot blot hybridization procedure for mrna quantitation in small samples of tissues...

ANALYTICAL BIOCHEMISTRY 172,436-443 (1988)

A Total Extract Dot Blot Hybridization Procedure for mRNA Quantitation in Small Samples of Tissues or Cultured Cells

ANDREW GRIMES, HARRY J. MCARDLE, AND JULIAN F. B. MERCER

Murdoch Institute,for Reseurch Into Birth D&s. Royal Childrens Hospitul. Flemington Road, Parkviile Victoria 3052, Australia

Received March 7, 1988

A simple method for the estimation of specific mRNA concentrations in small tissue samples (as little as 1 mg) or cultured cells (lower limit IO5 cells) is described. Guanidine hydrochloride extracts of whole cells or tissues are applied directly onto nitrocellulose and hybridized with the appropriate nick-translated probe. Loading according to DNA content allows expression ofthe result as concentration per cell. Hybridizing with a ribosomal RNA probe allows expression of results relative to rRNA and estimation of the RNA/DNA ratio in the sample. We describe the application of this procedure to the measurement of ceruloplasmin mRNA in tissues and cultured hepatocytes. CJ 1988 Academtc Press. Inc.

KEY WORDS: recombinant DNA technology; mRNA; RNA: DNA: gene expression.

Dot hybridization has proven very useful for analysis of mRNA levels and permits the analysis of many samples simultaneously (1). The method was made even more powerful by the development of the cytoplasmic dot blot method (2), which avoids the labor of RNA isolation. This procedure may be un- suitable for analysis of some tissue samples, however, because of ribonuclease destruction of the RNA during preparation of the cytoplasmic extracts (3, and A. Grimes, unpublished data). To avoid this problem, Cheley and Anderson (3) applied guanidine hydrochloride extracts of cultured cells to nitrocellulose and could detect viral nucleic acid sequences in the dots: however, they did not use the method for quantitation.

We have recently isolated a cDNA clone for rat ceruloplasmin (4) and our studies of the regulation of this gene involve analysis of many samples of both tissue and cultured cells. We describe a modification of the procedure of Cheley and Anderson (3), which allows rapid and accurate quantitation of specific mRNA sequences from a few milligrams of a liver biopsy or only IO5 cultured hepato-

cytes. Loadings on the filter are adjusted according to DNA content, and RNA content can be determined with an 18 S rRNA probe (5). Thus the results can be expressed as content of mRNA per cell, or per microgram RNA, and the RNA/DNA ratio can be calculated. Neither DNA nor protein in the sample interferes with the assay. This procedure is well suited to the analysis of mRNA levels in multiple small samples.

MATERIALS AND METHODS

Extraction procedures. Tissue extracts were prepared by homogenizing the sample in 10 vol of 7.5 M guanidine hydrochloride. 0.1% sarcosyl, 0.1 M 2-mercaptoethanol. 50 mM sodium citrate, pH 7 (Buffer A), at 4°C. This procedure was conveniently carried out using a Dounce homogenizer, although for some of the more fibrous tissues, a mechani- cal device. such as a Polytron homogenizer, was required. With biopsy samples or cell pel- lets, a larger volume to weight ratio was used, e.g., 200 gl of buffer for 3 mg of tissue. In these cases homogenization was carried out

OOO3-2697/88 $3.00 Copyright E 1988 by Academic Press. Inc All n&n of reprcduct~~ I” any form reserved.

436

TOTAL EXTRACT DOT BLOTS 437

using a disposable l-ml syringe with a 19- gauge needle to break up the clumps and then a 25gauge needle for complete homogenization. The extracts were centrifuged at 10,000 i-pm (9000g) in a Sorvall SS34 rotor for 10 min at 4°C to remove debris. A small aliquot was removed for determination of DNA content and the remainder stored at -20°C until required. We have found some evidence of breakdown of the RNA in this extract after 2 months at -20°C so for such long term stor- age -70°C is recommended.

DNA estimation. A modification of the procedure of Labarca and Paigen (6) was used. A portion of the extract (5 to 20 ~1) was added to 0.05 M Na phosphate, 2.0 M NaCl, pH 7.4, containing 1 pg/ml of Hoechst 33258 for standard extracts or 0.1 pg/ml for biopsy- sized samples. The fluorescence was mea- sured (excitation, 356 nm; emission, 458 nm) and the concentration of DNA in the extracts was determined by comparison with a standard curve, obtained using a range of concentrations of calf thymus DNA (0 to 15 pg for routine samples and 1 to 1.5 pg for biopsy- sized samples). The standard curve was not altered by the presence of 10 to 200 ~1 of the guanidine extraction buffer (results not shown).

Dot blot procedure. Extracts were diluted to a final concentration of 2 pg/ml DNA in prefiltered solution of 2.5 M formaldehyde in 6~ SSC’ (Buffer B, prefiltered through a 0.45-Km Durapor membrane), heated for 15 min at 65”C, cooled on ice, and centrifuged for 20 min at 10,000 i-pm (9000g) in a Sorvall SS34 rotor. This step not only denatures the RNA but also precipitates most of the protein and possibly DNA. Each sample was applied using gentle suction to a prewetted (water, then 10X SSC) nitrocellulose membrane (Schleicher & Schuell SRC-96). Serial dilutions of each sample were always used, nor- mally a range of 0. I to 0.6 pg of DNA. For hybridization with the ribosomal probe from 0.5 to 6 ng DNA was loaded (i.e., one 100th

’ SSC ( I X) is 0. I5 M NaCI, 0.0 15 M Na citrate, pH 8.

the level used for mRNA) although subse- quent experiments have shown that im- proved linearity is obtained if the loadings are one 10th of this (up to 0.6 ng DNA). The RNase A-treated control sample was prepared by diluting the guanidine extract 1:20 with water, adding the ribonuclease to a final concentration of 0.1 mg/ml, and incubating overnight at 37°C. DNase I or proteinase K was sometimes included at a concentration of 0.1 mg/ml. These treated extracts were then processed in the same way as the other extracts. Purified RNA was dotted in a range of 0.1 to 1 .O Fg for mRNA measurement and 2 to 10 ng for ribosomal RNA probing (as noted above, one 10th of this level may give better results). After application of the samples, the wells were washed with 500 ~1 of the Buffer B. The membrane was then air dried and baked for 120 min at 80°C in a vacuum oven.

Hybridization conditions. Filters were pre- hybridized with constant shaking for 2 h at 65°C in a plastic bag containing 25 ml of 6~ SSC, 10% dextran sulfate, 2X Denhardts (4 mg each of polyvinylpyrrolidone, Ficoll. and bovine serum albumin per ml), 2 IIIM EDTA, 0.1% (w/v) SDS,’ and 100 pg/ml of sheared and denatured herring sperm DNA. The pre- hybridization buffer was removed and mixed with the denatured probe and returned to the bag. Hybridization was for 18 h at 65°C. The filters were washed for 30 min each in the fol- lowing solutions: 4X SSC at room tempera- ture: 2X SSC, 0.1% SDS; 1 X SSC, 0.1% SDS; 0.5X SSC, 0.1% SDS; 0.2X SSC, 0.1% SDS, all at 65°C. The hybridization was checked by autoradiography using Kodak XAR-5 film. The dots were cut out and radioactivity was estimated in a Beckman liquid scintillation counter.

Probe labeling. A plasmid containing a 2- kb rat ceruloplasmin cDNA in pUC 18 (4)

* Abbreviations used: SDS, sodium dodecyl sulfate: DMM. Dulbecco’s minimal medium; CP, ceruloplasmin: EGTA, ethylene glycol bis(&aminoethyl ether) IV,A”-tetraacetic acid.

438 GRIMES. MC ARDLE. AND MERCER

was labeled to a specific activity of 2 X lo7 dpm/pg using a nick-translation kit supplied by Biotechnology Research Enterprises S.A. (Adelaide, South Australia). Unincorporated isotope was removed using gel filtration on Sephadex G-50. The 18 S rRNA probe (5) was kindly supplied by Dr. R. Guntaka, Uni- versity of Missouri at Columbia.

Hepatocyte culture. Hepatocytes were cultured according to the method of Page et al. (7). Briefly, a male Sprague-Dawley rat (250- 300 g) was injected intraperitonelly with Nembutal. The abdomen was opened, 5000 U heparin was injected into the inferior vena cava, and the portal vein was cannulated. The liver was perfused with 0.63 mM EGTA in wash buffer (5.4 mM KCl, 116 mM NaCl, 25 mM NaHC03, 5.5 mM glucose, 20 mM 4-(2- hydroxyethyl) - 1 - piperazineethanesulfonic acid, pH 7.4) followed by buffer without EGTA and buffer with 250 pg/ml collagenase (Boehringer-Mannheim) and 5 mM CaCl?. The cells were collected into Dulbecco’s minimal medium (DMM) containing 1% bovine serum albumin, washed three times, and plated onto 5-cm-diameter culture plates which had previously been coated with rat collagen. The cells were maintained for 4 h, washed, and cultured overnight in DMM containing 2% Ultroser G (LKB). The follow- ing day, the cells were washed with phosphate-buffered saline, scraped into the guanidine hydrochloride buffer, and processed in the same manner as the tissue samples.

RNA isolation. Total RNA was extracted from rat liver by the guanidine hydrochloride/CsCl ultracentrifuge method of Liu et al. (8) as modified by Wake and Mercer (9).

RESULTS

Guanidine hydrochloride extracts of the tissue or cells of interest were prepared and the loading onto nitrocellulose was adjusted according to the DNA concentration. In Fig. 1 we show the results of hybridizing the ceruloplasmin probe to a filter containing extracts of various tissues. Serial dilutions of the ex-

RNA Standards

02

0.4

0.6

0.8 B

1.0 *

0.2

0.4

0.6 *

0.8 %

1.0 e

RNase

RNase/DNase

RNase/Prot. K

*** Hepatocyte 2

9; * @ e + Hepaiocyte 1

Lung

H&l,,

Spleen

BraIn

Kldney

43 * Liver Lxopsy

i d LlV.3

0.05 0.1 0.2 0.3 0.4 0.5 0.6

pg DNA per Dot

FIG. 1. Total extract dot blots of various tissues hybridized with a rat ceruloplasmin probe. Guanidine hydrochloride extracts of the indicated tissues were prepared asdescribed under Materials and Methods, applied to nitrocellulose, and hybridized with a nick-translated rat ceruloplasmin probe (4). After hybridization. the filter was washed as described and exposed to X-ray film for 18 h at -7o’C with two intensifying screens. Samples were loaded according to DNA content; the amount of DNA in each dot is shown below the figure. At the top of the figure is shown extracts of liver diluted with water and treated by digestion overnight with RNase A alone (RNase). or RNase A and DNase (RNase/DNase) or RNase A followed by proteinase K (RNase/prot K) prior to application to the nitrocellulose. On the left-hand side of the filter are two sets of serial dilutions of purified rat liver RNA with the indicated amount (fig) of RNA per dot.

tracts were used, applying equivalent to 0.05 to 0.6 pg of DNA. The liver sample gave a clear signal that was detectable down to a loading of 0.05 pg of DNA. RNase treatment alone was sufficient to eliminate the signal, suggesting that DNA and protein were not contributing significantly to the result. The extracts from cultured hepatocytes gave in- tense signals, stronger with one preparation than the other. Correction for RNA loading, however, showed that the levels of CP mRNA in these cells were similar to those in liver extracts (see below). Signals from dots loaded with extracts oflung, heart, spleen, brain, and kidney were very faint and probably repre- sent nonspecific binding (see below).

The radioactivity in the dots was estimated


by scintillation counting and the results are presented in Fig. 2. The specific activity of the probe used was quite low (2 X 10’ dpm/pg) so the counts are low, yet are quite consistent over the range of DNA concentrations applied to the filter.

As expected from Fig. 1 the hepatocytes contained more CP mRNA per unit of DNA than the liver: hepatocyte preparation 1, 2- fold; preparation 2, 1.4-fold. The signal was proportional to DNA loading over the range used. The liver and liver biopsy results were essentially superimposed, showing that the accurate CP mRNA measurements can be made on a very small tissue sample. The counts from d’ots of other tissues gave signals that were less than 5 dpm above background at all DNA loadings. Only the results from spleen are shown in Fig. 2. The RNase treated. RNase + DNase, and RNase + proteinase K controls all gave background signals.

To express the mRNA levels relative to ribosomal RNA. loading rather than to DNA, an 18 S ribosomal RNA probe (5) was hybridized to dots of the same extracts. The loadings were one 100th that used for the CP mRNA.

160

140 .

.

. . 0 0

t.00 0.10 0.20 0.30 0.40 0.50 0.60

FIG. 2. Quantitation of CP mRNA per unit of DNA. Radioactivity in the dots was estimated by scintillation counting as described under Materials and Methods. Background counts based on radioactivity in parts ofthe filter not containing RNA were subtracted from the esti- mates. n , Hepatocyte 1; 0, hepatocyte 2: . . liver biopsy; e. liver; z!I. RNase A-treated control (liver): A, spleen,

600 r

. 500 k .

.

400 - II F I 0 P n . D 300 - 2

: 0

0 [r .

200 - q

8 . 0

DNA (ngidot)

FIG. 3. Quantitation ofthe rRNA content in the dotted extracts. The guanidine hydrochloride extracts were applied to nitrocellulose according to the DNA loadings indicated on the figure. Note that the loadings are one 100th those used for the ceruloplasmin mRNA measurements. The filter was hybridized with a nick-translated l&S rRNA probe (5) as described under Materials and Methods. Dots were excised and the radioactivity estimated by scintillation counting. The same method of background correction was used as in Fig. 2. n , Hepato- cyte I; 0. hepatocyte 2: ,:. liver biopsy; +, liver: A. spleen; A. RNase-treated control (liver).

Figure 3 shows the plot of the radioactivity in the dots according to DNA loading. The two hepatocyte extracts gave much higher signals than the other extracts. The two liver samples gave similar signals and the remaining tissues gave about one-half the signal of the liver samples (only the spleen is shown). RNase treatment reduced the signals to very low levels and there was no further reduction by ad- dition of a proteinase K digestion or DNase. Table I outlines the procedure for quantitat- ing the RNA loading but contains only two of the seven loading levels used in Fig. 3. A standard curve of dpm versus nanograms ribosomal RNA applied was obtained using dots containing calibrated amounts of purified RNA from liver (see “RNA-purified” in Table I). This allowed calculation of the amount of ribosomal RNA in the extracts. The standard curve was used to convert the counts in the samples into the RNA loadings (ng) shown in the third column.

These data can be used to estimate the

440 GRIMES. MC ARDLE, AND MERCER

3.6 212 6.3 1.75 1.85 2.1 172 5.0 1.85 3.6 1 0 3.6 2 0 3.6 I 0

1.8 245 7.36 4.09 3.16 0.9 124 3.42 3.80

3.6 99 2.6 0.12 0.69 2.1 78 1.9 0.71

315 10 265 8 229 6 149 4 86 2

TABLE I

EXAMPLEOFTHE DETERMINATION OFTHE RNA CONTENT IN THE SAMPLES

Sample DNA (ng)” RNA (dpm)’ RNA (ng)’ RNA/DNAd RNA/DNA (Av.)’

Liver

RNase’ RNase/prot K’ RNase/DNase/

Hepatocyte 1

Kidney

RNA (purified~

-.

’ This is the amount of DNA (ng) in the sample loaded on the dot determined by the Hoechst method (6). Each sample was loaded at seven levels. only two of which are shown here.

’ Radioactivity in the dot after hybridization with a nick-translated 18 S rRNA probe (5) corrected by subtracting the background which was the average of six dots not containing RNA.

“The ribosomal RNA content of the dot is calculated from a standard curve constructed from the counts obtained with the purified rat liver RNA.

‘Calculated from this data and should be regarded as relative since it depends on the purity of the standard RNA. Accurate values for all the samples can be obtained by using the RNA/DNA ratio of one tissue determined by an independent procedure.

’ Average of all the measurements carried out with this sample, some of which are not shown. ’ Liver samples digested with RNase A, alone or together with DNase or proteinase K as described under Materials

and Methods. y RNA isolated from rat liver using the guanidine hydrochloride procedure (8,9).

RNA/DNA ratios in the various tissues. The hepatocyte preparation 1 had an average RNA/DNA ratio of 3.74 compared with 1.85 for the liver, i.e., about two-fold higher; the other tissues all had RNA/DNA ratios around 0.7. These RNA/DNA ratios are useful for comparison between samples, but the values are lower than those estimated from other studies. For example, using the procedure of Swift et al. (10) we have found that the RNA/DNA ratio in adult male rat liver is 2.2 rather than 1.85 (S. A. Wake and J. F. B. Mercer, unpublished data). The discrepancy is possibly due to the loss of tRNA during the ethanol precipation of the RNA standard from the guanidine hydrochloride solution. Corrected values of the RNA/DNA ratio in

the other tissues is obtained using the value of 2.2 for the liver and adjusting the other samples relative to this. Using this procedure we estimate the corrected RNA/DNA ratios in the samples to be kidney, 0.83; brain, 0.77; spleen, 0.68; heart, 0.71; lung, 0.86; hepatocyte 1,4.5; and hepatocyte 2, 3.2.

Table 2 shows how the data can now be expressed relative to DNA or rRNA. The dpm obtained with the ceruloplasmin probe are shown in the third column, and dpm per microgram DNA are shown in the fourth column. If a suitable CP mRNA standard were included with the dots [e.g., an SP6 transcript (1 l)], this data could be converted to molecules/cell. Using the RNA/DNA ratios from Table 1, the amounts of RNA for each DNA


TABLE 2

EXAMPLE OF THE DETERMINATION OF THE CERULOPLASMIN mRNA CONTENT IN THE SAMPLES

Sample DNA bid CP mRNA (dpm)” CP mRNA

dpm/pg DNA’ RNA (/.eY’ CP mRNA

dpm/pg RNA’

Liver 0.45 52 116 0.83 62 0.36 36 100 0.67 54

RNas$ 0.36 -2 0 0 - RNase/prot K’ 0.36 -1 0 0 RNase/DNase’ 0.36 2 5x 0 -

Hepatocyte 1 0.45 114 253 1.7 67 0.36 86 238 1.3 66

Kidney 0.45 2” 4x 0.31 6” 0.36 2” 58 0.25 8R

RNA (purified) 60 0.8 75 36 0.6 60

” Levels determined by the Hoechst method (6); note that loadings are in micrograms for the CP mRNA determination, but in nanograms for rRNA (Table I). Each sample was loaded at seven levels (0.045-0.54 pg): only two are shown here.

h Determined by hybridization with a nick-translated rat ceruloplasmin probe. The background (45 dpm) was determined for two dots not containing RNA and has been subtracted.

” Plotted in full in Fig. 2. *Determined using the DNA loading from column 1 and average RNA/DNA ratio from Table I. ‘Plotted in full in Fig. 4. ‘Liver RNA digested with RNase, alone or together with proteinase K or DNase as described under Materials and

Methods. x These values deo not differ significantly from background.

loading can be estimated (5th column), and hence the dpm of CP mRNA per microgram RNA can be obtained (6th column). When expressed as CR mRNA per cell, the levels in cultured hepatocytes are about double those in liver extracts. Expressed per unit of RNA, the levels of CP mRNA are the same in liver and hepatocytes. Figure 4 shows some of the data from Fig. 2 expressed relative to RNA loading, and there is no difference among the hepatocyte 1, hepatocyte 2, liver, or liver biopsy sample.

DISCUSSION

We have found the procedure described here to be very useful for the rapid analysis of mRNA levels in multiple samples and it is applicable particularly to experiments where

the amount of sample is limiting, as when using cultured cells. Other workers have used the cytoplasmic dot blot procedure of White and Bancroft (2) for RNA analysis of cultured cells. We believe our procedure is more reliable because it removes the risk of nuclease digestion, a problem which we found to com- plicate the use of cytoplasmic dots for analysis of tissue samples.

One of the important features of the method is that genomic DNA is not removed, allowing the DNA concentration to be used to determine the loading on the filter. The DNA apparently did not interfere with measurements of mRNA, since RNase and RNase + DNase controls gave identical results. It is possible that much of the DNA is removed by the heat treatment in Buffer B, but we have not investigated this.

442 GRIMES, MC ARDLE, AND MERCER

150

E 100 D

2 E

8 50

.

.

Om+AA"""A I I I I 0.0 0.5 1 .o 1.5 2.0 2.5 3.0

RNA (ugidor)

FIG. 4. Correction of CP mRNA against rRNA. Using the data from Fig. 2 and Fig. 3. the radioactivity due to the CP mRNA was plotted against the amount of rRNA applied to the filter. n , Hepatocyte 1: Cl, hepatocyte 2; c.,‘, liver biopsy; +. liver: A, spleen: a. RNase-treated control (liver).

The method is suitable for analysis of small tissue samples, for example, samples obtained by needle biopsy of human liver. Anal- ysis of 3 mg of liver gave results identical to those obtained with several hundred milligrams. The standard guanidine hydrochloride procedure for RNA isolation (8,9) is not readily applicable to such small samples. We have also used the method to analyze the expression of collagen type IV mRNA in different tissues during development of mice (results not shown).

Our procedure allows the data to be expressed relative to DNA or rRNA content. This would permit the calculation of the number of mRNA molecules per cell or per microgram RNA, if a CP mRNA standard generated using an SP6 transcription system were used (11). The value of obtaining esti- mates based on rRNA or DNA content was illustrated by comparing the results obtained in hepatocytes in culture and in the liver extracts. On a DNA basis (i.e., molecules/cell), the hepatocytes contained more CP mRNA than the liver, but when expressed relative to rRNA, the levels of RNA were the same. The explanation for this is presumably that the

liver contains a mixed population of cells, whereas the hepatocytes isolation procedure is designed to remove nonhepatic cells and should reflect the true hepatocyte levels. The nonhepatic cells in the liver must have low RNA/DNA ratios, thus reducing the CP mRNA/cell in extracts of total liver.

We used relatively low specific activity probes in this work, and the counts obtained were quite low, yet were clearly reliable since the data showed linearity with respect to DNA loading even when the signal was below 20 dpm. The low counts did limit the inter- pretation of the results obtained with extracts of lung, heart, spleen, brain, and kidney. We were able to determine only that the levels of CP mRNA in these other tissues were less than 10% of the level in the liver. We suspect, however, that the CP gene is not expressed in these tissues. Expression has been demon- strated in the choroid plexus (4) but this comprises a very small part of the whole brain. It may be possible to improve the de- tection limit by using a probe of higher specific activity, but the limitation may be the background signal on the filter arising from residual DNA or protein. Another modification of the procedure which we are currently investigating is the use of a slot blot apparatus and densitometry to allow more rapid data collection.

In summary, we have described a method for analysis of mRNA levels which is rapid and applicable to the analysis of small samples. In this paper we discuss only the analysis of CP mRNA, however, in our laboratory we have also used the method for measurement of metallothionein, phenylalanine hydroxy- lase, and collagen type IV mRNAs and we consider that it should prove to be generally applicable.

ACKNOWLEDGMENTS

We thank David Danks for his advice and criticism of the manuscript. This work was supported in part by the Australian National Health and Medical Research Council.


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2. White. B. A., and Bancroft, F. C. (1982) J. Biol. Ctwn. 257, 8569-8572.

3. Cheley, S.. and Anderson. R. (1984) Anal. Bioclwr~~. 137,15-19.

4. Aldred, A., Grimes. A., Schreiber. G.. and Mercer. J. F. B. (1987) J. Biol. C/WH. 262,2875-2878.

5. Katz, R. A., Erlanger. B. F.. and Guntaka, R. V. ( 1983) Biochim. BiophJs. .kra 739, 258-264.

6. Labarca C., and Paigen, K. ( 1980) Anal. Biochern. 102,344-352.

7. Page. M. A., Baker, E.. and Morgan, E. H. (1984) .4rner J. PIq:C)l. 246. G26-G33.

8. Liu, C.-P.. Slate, 0. L.. Cravel. R.. and Ruddle, F. M. (1979) Proc. Null. .-icad. Sri. USA 76.4503-4506.

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10. Swift. G. H., Hammer, R. E., MacDonald, R. J.. and Brinster. R. L. ( 1984) c’c~ll38,639-646.

I I. Melton. 1). A., Krieg, P. A., Rebagliati, M. R.. Mani- atis. T.. Zinn, K., and Green, M. K. (1984) Nuclm.kids Rer. 12, 7035-7056.

a total extract dot blot hybridization procedure for mrna quantitation in small samples of tissues...

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