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Technical

A Simple and A situation c o m m o n l y encountered in

the laboratory is one in w h i c h com-
50 mM KCI, 10 mM Tris-HCl (pH 8.3),
2.5 ~M r a n d o m hexamers (Pharmacia),
Rapid Method to parison of the relative levels of expres- 1 mM each dNTP (Promega), 1 U/~l
sion of specific genes in h i g h l y purified RNase inhibitor, and 2.5 U/~l Moloney
Analyze Specific populations of cells or in clinical m u r i n e leukemia virus (Mo-MLV)
mRNAs from Few samples would be very useful but is
problematic due to the small a m o u n t
reverse transcriptase (GIBCO, BRL). A
master mix of all reagents i n c l u d i n g
Cells in a of material available. Therefore, we the e n z y m e is prepared on ice and 17
Semi-quantitative have devised a simple and rapid meth-
od to analyze in a semi-quantitative
~l is added to each RNA sample. The
samples are then incubated 10 rain at
Way Using the way mRNAs from relatively few cells
using the polymerase chain reaction
room temperature followed by 45 m i n
at 42°C and 5 rain at 95°C. The
Polymerase Chain (PCR). This m e t h o d is particularly use- samples can t h e n be frozen or directly
Reaction ful for rapid determination of the ef-
fect of differentiative agents, cytokines,
amplified.
PCR is performed in 2 mM MgCl2,
antisense oligonucleotides, or pharma- 50 mM KC1, 10 mM Tris-HC1 (pH 8.3),
ceutical agents on the expression of 0.2 mM each dNTP, and 2.5 U/100 ~l
J. Golay, F. Passerini, specific genes in a semi-quantitative Taq DNA polymerase (Perkin-Elmer
and M. Introna way. It could also be used to detect and Cetus) and with 5 ng/~l of each
quantify m i n i m a l residual disease specific primer. As for the reverse tran-
Istituto Ricerche Farmacologiche Mario where few cells are available for study. scriptase reaction, a master mix of all
Negri, 20157 Milan, Italy PCR reagents including the enzyme is
METHOD prepared and equally distributed in the
We tested several published methods different reaction tubes. An aliquot of
for isolating RNA from few cells and the products of the reverse transcrip-
found that the best m e t h o d for our tase reaction (1-5 ~l) is t h e n added to
purpose was by guanidine thiocyanate each tube. Comparison of the levels of
and a modified acid phenol extrac- gene expression between different
tion. I11 This m e t h o d gives undegraded samples requires that the PCR reaction
RNA and is highly reproducible in its be stopped during the exponential
efficiency of both RNA extraction and phase of amplification. This can easily
subsequent reverse transcription, fac- be d e t e r m i n e d by removing an aliquot
tors that are important for adequate at regular intervals during the PCR.
quantification. The m e t h o d described With further experience of the behav-
below works well for 1-10 x 103 cells in ior of the particular gene under study,
the case of hematopoietic lines and 10 an aliquot can be taken out at only
times that n u m b e r in the case of two different times (e.g., after 22 and
normal lymphocytes. 25 cycles) to ensure that all samples are
Briefly, the cells are washed once in still w i t h i n the exponential phase of
sterile saline and the pellet is re- amplification. In addition, to measure
suspended by vortexing in 150 ~l of the efficiency of RNA extraction and of
guanidine thiocyanate solution (4.2 M) the reverse transcriptase reaction for
c o n t a i n i n g 0.5% sodium lauryl sar- each sample, the [5-actin gene is
cosinate, 25 mM sodium citrate (pH 7), amplified from a 1- to 2-~1 aliquot of
100 mM 2-mercaptoethanol, and 1 ~g the same reverse transcription reaction
of E. coli RNA. To this are added 4.5 ~l and used as an internal standard.
of 2 M sodium acetate (pH 4), 180 ~l of All PCR products are run in an
water-saturated phenol, and 35 ~l agarose gel and blotted onto nitrocel-
chloroform. The samples are vortexed, lulose filters, w h i c h are then hybrid-
put on ice 10 min, and then centri- ized with the appropriate 32p-labeled
fuged at 12,000 g for 10 m i n at 4°C. plasmid probes. The signals obtained
Next, 80 [xl of the water phase is care- are quantified by densitometric analy-
fully transferred to another Eppendorf, sis on an image analyzer (Research
2.7 volumes of ethanol is added, and Analysis System R-1000, Amersham).
the RNA is i m m e d i a t e l y precipitated by
centrifuging at 12,000 g for 20 rain at EXAMPLE
4°C. The pellet is washed in cold 70% As a test gene, we chose the B-myb
ethanol, vacuum-dried, and dissolved gene, w h i c h is expressed to an average
in 3 ~l of H20. This RNA is directly level by the HL-60 cell line under
reversed-transcribed in 5 mM MgC12, n o r m a l conditions but becomes vir-

144 PCR Methods and Applications 1:144-145©1991 by Cold Spring Harbor Laboratory Press ISSN 10.54-9803/91 $3.00
 Downloaded from genome.cshlp.org on April 30, 2019 - Published by Cold Spring Harbor Laboratory Press

Blllllll! Technical Tips

tually u n d e t e c t a b l e after s t i m u l a t i o n of 2. E x t r a p o l a t i o n of the values o b t a i n e d

these cells for 48 hr by p h o r b o l esters for the HL-60 samples before a n d after
(PMA), as d e t e r m i n e d by N o r t h e r n blot t r e a t m e n t with PMA s h o w t h a t PMA
analysis (Fig. 1, u p p e r panel). RNA reduces B-myb mRNA levels by 9 0 - 9 5 %
from 2 x 103 HL-60 cells before a n d (Fig. 2), w h i c h is in reasonable agree-
after t r e a t m e n t w i t h PMA for 48 hr was m e n t w i t h the N o r t h e r n data (Fig. 1).
extracted in duplicate, reverse-tran-
scribed, a n d amplified w i t h t h e ap- ACKNOWLEDGMENTS
propriate primers u n d e r t h e c o n d i t i o n s This work was s u p p o r t e d by the Italian
i n d i c a t e d in Figure 1. The results (Fig. Association for C a n c e r Research, t h e
1, lower panel) d e m o n s t r a t e t h a t t h e F o u n d a t i o n T e t t a m a n t i , a n d a Fellow-
strong r e d u c t i o n in B-myb gene expres- ship from t h e C o m m i s s i o n of the Euro-
sion after PMA t r e a t m e n t is also detec- p e a n C o m m u n i t y to J.G. We t h a n k Dr.
table by PCR analysis, a n d to a level S. Haskil a n d S. Becker for useful sug-
e q u i v a l e n t to t h a t o b t a i n e d by N o r t h - gestions.
ern analysis. The levels of 13-actin
m e a s u r e d by PCR are, o n the o t h e r REFERENCES
h a n d , u n c h a n g e d after PMA t r e a t m e n t . 1. Belyavsky, A., T. Vinogradova, and K.
The results also s h o w t h a t the m e t h o d Rajevsky. 1989. PCR-based cDNA
of RNA extraction, reverse transcrip- library construction: General cDNA
tion, a n d PCR gives g o o d reproduc- libraries at the level of a few cells.
ibility because t h e i n t e n s i t y of t h e NucleicAcids Res. 17. 2919-2932.
b a n d s from duplicate samples differs 2. Chelly, J., J.C. Kaplan, P. Maire, S.
by n o m o r e t h a n 15% as d e t e r m i n e d Gautran, and A. Kahn. 1988.
by d e n s i t o m e t r i c analysis. Transcription of the dystrophin gene
It is i m p o r t a n t to n o t e t h a t this in human muscle and non,muscle
m e t h o d also p e r m i t s q u a n t i f i c a t i o n of tissues. Nature333; 858-860.
relative levels of specific mRNAs by 3. Golay, J., A. Capucci, M. Arsura, M.
FIGURE 1 Comparison of B-myb gene levels c o n s t r u c t i n g a s t a n d a r d curve using Castellano, V. Rizzo, and M. Introna.
measured in Northern blots or by PCR. HL- serial d i l u t i o n s of RNA c o n t a i n i n g t h e 1991. Expression of c-myb, but notA-
60 cells were stimulated for 48 hr with l0 gene of interest a m p l i f i e d in parallel to myb, correlates with proliferation in
ng/ml of PMA. Total RNA was extracted in t h e u n k n o w n samples, as described in human hematopoietic cells. Blood 7;
guanidium thiocyanate solution and m o r e detail in previous publications. (2) 149-158.
purified on cesium chloride gradients. (3) The i n t e n s i t y of each b a n d is m e a s u r e d
Levels of B.myb mRNA before (lane a) and by d e n s i t o m e t r i c analysis a n d plotted. Received July 8, 1991; accepted in revised
after (lane b) PMA treatment were
determined by standard Northern analysis
An e x a m p l e of such a s t a n d a r d curve form September 12, 1991.
for the B-myb gene is s h o w n in Figure
(upper panel). Alternatively, the micro-
method of RNA extraction was performed as
described in duplicate on 2 × 103 HL-60 Standard curve
cells, before and after treatment with PMA 1000
for 48 hr (lowerpanel, lanes a and b, respec-
tively). Each sample was then reverse- u~
transcribed in a total volume of 20 ~1. For B- -PMA
o
myb amplification, a 5-~1 aliquot from each 100
reverse-transcription reaction was then m

amplified in a 25-~1 volume for 24 cycles as Z -n

follows: 1 min at 95°C, 2 rain at 64°C, and
2 min 30 sec at 72°C. The primers E~ 10 +PMA
dATGTCTCGGCGGACGCGCTGCGAG and
•-~ :5
dGCCGTCCTTGTCCTCGAGCTCCAGC
amplify a fragment of 633 bases. For 13-actin • v I
amplification, 2-~tl aliquots were amplified n-
I
by PCR for 22 cycles of 1 rain each at 95°C, 1 " • • " .... I • " • ' ' ' " ! I ° ''°°'l • . . . . ,"

55°C, and 72°C. The primers ,01 ,1 1 10 100

dCCTTCCTGGGCATGGAGTCCTG and ng HL60 RNA
dGGAGCAATGATCTTGATCTTC amplify a FIGURE 2 Standard curve for B-mybamplification. A total of ] 0 0 ng HL-60 RNA was reverse-
fragment of 202 bases. A 10-~1 aliquot of transcribed in 20 ~tl, and a 2-~tl aliquot and twofold dilution of it were amplified using the B-
each reaction product was run in a 1.5% myb primers as described in Fig. 1. The specific hybridization signals obtained were
agarose gel, transferred to nitrocellulose, quantified by densitometric analysis and plotted. The average values for the signals obtained
and hybridized with the appropriate 32p. with HL-60 cells before and after treatment with PMA (396 and 10, respectively) are also indi-
labeled plasmid probes as described else- cated with dashed lines, showing that PMA reduces the levels of B-myb mRNA by 90-95%
where.(3) (from 3 to 0.23 ng equivalent of HL-60 RNA).

PCR Methods and Applications 145

 Downloaded from genome.cshlp.org on April 30, 2019 - Published by Cold Spring Harbor Laboratory Press

A simple and rapid method to analyze specific mRNAs from few

cells in a semi-quantitative way using the polymerase chain
reaction.
J Golay, F Passerini and M Introna

Genome Res. 1991 1: 144-145

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