 |
||||
| 2007 JASN IMPACT FACTOR 7.111 | HOME AUTHOR INFO EDITORIAL BOARD SUBSCRIBE FEEDBACK ALERTS HELP | |||
| CURRENT ISSUE | ARCHIVES | JASN Express | ONLINE SUBMISSION | |
|
||||
| 2007 JASN IMPACT FACTOR 7.111 | HOME AUTHOR INFO EDITORIAL BOARD SUBSCRIBE FEEDBACK ALERTS HELP | |||
| CURRENT ISSUE | ARCHIVES | JASN Express | ONLINE SUBMISSION | |
REGULAR ARTICLES |
Department of Pathology, Leiden University Medical Center, Leiden, The Netherlands.
Correspondence to Michael Eikmans, Leiden University Medical Center, Department of Pathology, P. O. Box 9600, Building 1, L1-Q, 2300 RC Leiden, The Netherlands. Phone: +31 71 5266625; Fax: +31 71 5248158; E-mail: MEikmans{at}Pat.AZL.NL
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
1(IV) mRNA was analyzed as a
measure for RNA integrity. The results show that at least 3 h may pass between
microdissecting the renal tissue and the onset of cDNA synthesis without
degradation of the glomerular mRNA. To extract the glomerular mRNA,
microdissected glomeruli were incubated in a permeabilization solution.
Treating glomeruli with collagenase IV before permeabilization had a
deteriorating effect on the mRNA yield. The addition of reverse transcription
mixture to the permeabilization solution in the presence of the glomeruli
resulted in the highest cDNA yields. Storage of glomerular tissue in the
presence of Nonidet P-40-based buffer for 1 wk at -70°C did not
significantly affect the mRNA, but storage for 2 or 4 wk resulted in
deterioration of the mRNA by approximately 40 and 95%, respectively.
Furthermore, three methods for total RNA isolation from microdissected
interstitial tissue were compared. An approximately 2.5 times higher yield of
collagen 1(IV) mRNA was obtained with silica gel-based membrane spin
technology than with a guanidine isothiocyanate/phenol chloroform or a lithium
chloride/phenol chloroform method. Finally, this study shows for the first
time reliable detection of collagen 1(IV) mRNA in biopsies that had
been frozen for at least 10 yr at -70°C. These experiments have helped to
improve a procedure for the processing of glomerular and interstitial tissue
acquired from human kidney biopsies for mRNA analysis. This method is suitable
for implementation in routine clinical practice. |
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Before investigating the clinical implications of ECM mRNA extraction in
renal biopsies, it is necessary to optimize the steps that are involved in
extracting the RNA from the tissue. In the investigation reported here, we
improved a method for extraction and storage of minute quantities of RNA from
glomerular and interstitial tissue, analyzing mRNA levels of the collagen
1(IV) molecule as a measure for RNA integrity. We show that we could
detect collagen 1(IV) mRNA levels in biopsies that had been frozen up
to 10 yr.
This method allows the quantification of mRNA levels for molecules involved in renal pathology, such as ECM molecules and cytokines, in clinically obtained human kidney biopsies.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
To investigate whether glomeruli were left in the interstitial fraction after microdissection, or, alternatively, whether interstitial components were carried over to the glomerular fraction, two pieces of cortical tissue 2 mm long were microdissected as described previously. Glomerular and interstitial tissues were separately pelleted by centrifugation, and fixed overnight in paraformaldehyde. After embedding in plastic, the tissues were completely cut up in sections and stained with toluidine blue for light microscopic analysis.
RNA Extraction from Glomerular Tissue
For these experiments, we used glomeruli obtained by microdissection of
each of the three cadaveric donor kidneys, and compared several methods,
described in the literature
(10,12,15),
suitable for RNA extraction from minute quantities of glomerular tissue. We
used reverse transcription (RT)-PCR for amplification of the collagen
1(IV) mRNA present in the glomerular samples as a measure for RNA
yield. Experiments were performed in duplicate.
From each kidney, eight batches of 10 glomeruli were pelleted by centrifugation for 1 min at 12,000 x g at room temperature to remove PBS. To investigate whether treatment of glomeruli with collagenase IV after microdissection but before permeabilization improves glomerular RNA extraction, four batches were incubated with 5 U of collagenase IV (Sigma, St. Louis, MO; catalog no. C1889) containing 40 U of rRNasin for 15 min at 37°C. As controls, four batches were incubated with PBS containing 40 U of rRNasin under the same conditions. The collagenase IV-treated glomeruli were washed once with 20 µl of PBS to remove the collagenase IV. We compared Nonidet P-40- and Triton X-100-based solutions for their ability to permeabilize glomerular cells. From each of the four batches, two were incubated in Nonidet P-40-based buffer (0.5% Nonidet P-40, 10 mM Tris buffer at pH 8.0, 10 mM NaCl, and 3 mM MgCl2, supplemented with 1 U/µ1 rRNasin) and two in Triton X-100-based solution (0.9% Triton X-100, 5 mM dithiothreitol, and 1 U/µl rRNasin) on ice for 5 min. The glomeruli were resuspended in a reaction volume of 10 µl by gently tapping the reaction tube.
For conversion of glomerular mRNA into cDNA, we used a mixture containing 5 U of avian myeloblastosis virus-reverse transcriptase (Boehringer Mannheim), 1 µM oligo(dT)15 (Boehringer Mannheim), 2 mM dNTP, and 10 U of rRNasin (Promega) in 1x RT buffer (Boehringer Mannheim). We compared two conditions of cDNA synthesis to find out which resulted in the highest cDNA yield: addition of RT mixture to the permeabilization solution either directly or after removal of the permeabilized glomeruli from this solution by centrifugation for 1 min at 12,000 x g at room temperature. The RT reactions were carried out for 1 h at 37°C and subsequently boiled for 5 min. Negative RT controls, using a solution containing all components except for the reverse transcriptase enzyme, were performed to check for contamination of the cDNA samples by genomic DNA.
Storage of Glomerular Tissue
To test the effect of storage time of glomerular tissue on the quality of
the mRNA, eight batches containing 10 non-collagenase IV-treated glomeruli
from each of the three donor kidneys were incubated either in 10 µl of
Nonidet P-40 buffer, in 10 µl of Triton X-100 solution, both supplemented
with 60 U of rRNasin, or in 3 µl of rRNasin (60 U) alone, and were
immediately frozen on CO2 ice. Under each condition, two batches
(duplicates) were stored, either for 1 wk, 2 wk, or 4 wk, at -70°C. Two
batches were frozen and immediately defrosted, and were used as controls.
Afterward, the solutions containing the glomeruli were thawed on ice, and the
glomerular cells were permeabilized in the solution they had been stored in.
Nonidet P-40 buffer was used for glomerular batches that had been stored in
rRNasin.
RNA Extraction from Interstitial Tissue
Six 16-gauge needle biopsies were taken from a cadaveric donor kidney.
Cortical tissue samples approximately 2 mm long were microdissected as
described above. Interstitial tissues were pelleted by centrifugation to
remove the PBS and then weighed. Total RNA was isolated from the interstitial
tissue either by silica gel-based membrane spin (SGMS) technology (RNeasy®
mini kit; Qiagen, Chatsworth, CA), guanidine isothiocyanate/phenol chloroform
(GITC/PC; TRIzol® from Life Technologies BRL), or a lithium
chloride/phenol chloroform (LiCl/PC) procedure. Weights of the tissues used
for each of the three different protocols were 5.4 and 5.8 mg, 5.6 and 5.4 mg,
and 7 and 6.8 mg, respectively. Interstitial tissues were resuspended, in
duplicate, in 350 µl of RNeasy® lysis buffer, 500 µl of TRIzol®
solution, or 500 µl of 3 M LiCl/6 M urea solution, respectively, and mixed
rigorously (Ultra-Turrax; Janke & Kunkel, Zoetermeer, The Netherlands) for
45 s. The SGMS and GITC/PC methods were performed according to the supplier's
manual. The LiCl/PC method was performed according to an earlier report
(17). For complete RNA
precipitation with the Li/PC and the GITC/PC method, 5 µg of glycogen
(Boehringer Mannheim) was added. The RNA obtained with the three RNA isolation
methods was dissolved in equal amounts of RNase-free water (56 µl). Ten
microliters of each RNA sample was incubated for 15 min at 60°C, and cDNA
was synthesized by adding 10 µl of RT mixture. As a measure for RNA yield
obtained with each of the three extraction methods, the collagen 1(IV)
mRNA yield per milligram of tissue was measured by competitive RT-PCR.
Storage of Nonmicrodissected Biopsy Tissue
Biopsy specimens used in this experiment were obtained from patients with
IgA nephropathy. Informed consent was given by the patients for use of part of
the biopsy for scientific purposes. Biopsies were taken with an 18-gauge
needle.
We used five biopsies that had been stored for different periods of time at
-70°C (between 1 and 10 yr). After assessing the location of the cortex in
each biopsy by light microscopy, a piece of cortical tissue 1 mm long was cut
off. RNA was isolated with the SGMS method, and collagen 1(IV) cDNA was
measured by RT-PCR in triplicate.
PCR Conditions
Primers. To distinguish PCR amplification of cDNA from that of
genomic DNA by the size of the amplified products, human collagen 1(IV)
primers (Life Technologies BRL) were localized to separate exons, spanning
three introns. Collagen 1(IV) primer sequences were sense 5'-ATG
TCA ATG GCA CCC ATC AC-3' and antisense 5'-CTT CAA GGT GGA CGG CGT
AG-3', yielding an amplified product of 382 bp as predicted from the
cDNA sequence. The primers were designed in the coding region of the NC1
domain of the collagen 1(IV) molecule, and are located approximately
300 bp upstream of the poly(A) tail.
PCR Assay. Glomerular collagen 1(IV) cDNA levels obtained in
the experiments with the three cadaveric donor kidneys were semiquantitatively
assessed by PCR. Each amplification reaction contained an amount of cDNA
equivalent to that obtained from one-half of a glomerulus (i.e., 1
µl of the cDNA sample). The amplification mixture contained 1 µM of both
primers, 0.2 mM dNTP, 2 mM Mg2+, 1 U of AmpliTaq, and 1x
AmpliTaq buffer (Perkin-Elmer Cetus) in a total volume of 25 µl. The first
incubation in the thermal cycler (Perkin-Elmer Cetus) was performed at
94°C for 7 min. This was followed by 35 cycles consisting of the following
steps: 94°C for 1 min, 65°C for 0.5 min, and 72°C for 1.5 min. The
final incubation was performed at 72°C for 7 min. All PCR assays were in
exponential phase.
Analysis of the PCR Products. The amplified products were separated by agarose gel electrophoresis. Eight microliters of the reaction mixture was applied to a 1.5% agarose gel in Tris Borate/ethylenediaminetetra-acetic acid buffer. DNA bands were visualized by ethidium bromide staining and ultraviolet transillumination. The densitometric values of the amplified cDNA bands were calculated by densitometric image scanning (hardware used was The Imager from Molecular Dynamics; software was Appligene, Inc., version 2.03, and ImageQuant, version 4.2a).
Statistical Analyses
Data are presented as means ± SD. Paired t tests were used
for statistical analysis of differences in PCR signals. P < 0.05
was considered statistically significant.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
We found that with 3 h between the onset of microdissection performed in
ice-cold PBS and the start of cDNA synthesis, the relative glomerular collagen
1(IV) PCR signal was not significantly lower compared with glomerular
tissue that was immediately processed for cDNA synthesis after microdissection
(data not shown), indicating that the mRNA had not been degraded.
RNA Extraction from Glomerular Tissue
Figure 1 shows that the
relative cDNA yield obtained from glomeruli treated with collagenase IV (0.16
± 0.02 and 0.11 ± 0.05; bars 5 and 6) was significantly lower
than that from nontreated glomeruli (1 ± 0.17 and 0.99 ± 0.04;
bars 1 and 2; P < 0.01). Application of Nonidet P-40 or Triton
X-100 solution for permeabilization of glomerular samples gives no significant
difference in the efficiency of mRNA extraction. The relative amplified
glomerular cDNA signals for Nonidet P-40- and Triton X-100-treated samples
were 1 ± 0.17 (bar 1) and 0.99 ± 0.04 (bar 2; P = NS),
respectively. Finally, Figure 1
shows that addition of RT mixture to the permeabilization solution in the
presence of the glomerular cells results in a significantly higher collagen
1(IV) cDNA yield (1 ± 0.17 and 0.99 ± 0.04; bars 1 and 2)
than with the glomerular cells removed (0.46 ± 0.15 and 0.45 ±
0.23; bars 3 and 4; P < 0.05).
|
Storage of Glomerular Tissue
Figure 2 shows that storage
of glomeruli for 1 wk at -70°C in the presence of Triton X-100 solution
(0.71 ± 0.06) or rRNasin alone (0.66 ± 0.17) resulted in a
significant decrease of the collagen 1(IV) signal compared to glomeruli
that had been frozen under the same conditions but immediately defrosted (1
± 0.09 and 1 ± 0.17; P < 0.01). However, storage in
the presence of Nonidet P-40 buffer for 1 wk did not result in a significant
decrease (0.84 ± 0.21 versus 1 ± 0.13; P =
NS). After 2 wk of storage in Nonidet P-40, Triton X-100, or rRNasin alone,
relative levels had significantly decreased to 0.61 ± 0.19, 0.46
± 0.22, and 0.37 ± 0.21, respectively (P < 0.05).
After 4 wk, relative levels were 0.07 ± 0.05, 0.06 ± 0.04, and
0.05 ± 0.04, respectively (P < 0.01).
|
RNA Extraction from Interstitial Tissue
We found that the yield of interstitial collagen 1(IV) mRNA per
milligram of tissue obtained with SGMS, measured by assessment of the yield of
the corresponding cDNA with the aid of competitive PCR, was at least 2.5 times
higher than that obtained with GITC/PC or LiCl/PC (data not shown).
Amplification of negative RT controls for all samples did not show any
nonspecific bands, indicating that there was no contamination of genomic
DNA.
Storage of Nonmicrodissected Biopsy Tissue
In previous experiments (unpublished), we found that it was not possible to
obtain intact RNA from microdissected tissue that had been frozen and
defrosted before microdissection. Therefore, mRNA levels in frozen biopsies
could only be analyzed in nondissected, total cortical tissue. Collagen
1(IV) mRNA was measured with RT-PCR in biopsies that had been frozen
for 1, 3, 5, or 10 yr at -70°C (Figure
3). Relative levels were 1 ± 0.16, 0.62 ± 0.17, 0.63
± 0.12, and 0.81 ± 0.07, respectively. The mRNA did not
significantly deteriorate between 1 and 10 yr of storage.
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
We analyzed mRNA levels for collagen 1(IV) by RT-PCR as a measure
for efficiency of RNA extraction and for degradation of mRNA. One of our
motives for looking at collagen 1(IV) is that it is a low abundance
gene. When it is possible to detect such a transcript, it is likely that
higher abundant transcripts certainly can be picked up. Another reason for our
focus on collagen 1(IV) is that its mRNA has been shown to be
upregulated in diseased and sclerotic renal tissue
(1,6,10,11),
suggesting a potential role in renal pathology.
We chose PCR primers that anneal to the collagen 1(IV) cDNA very
near to the poly(A) tail of its corresponding mRNA, the starting site for the
cDNA synthesis. In that way we sought to circumvent the inefficiency of the RT
reaction and amplify as much cDNA as possible containing both primer sites. We
believe that if one wished to perform an amplification using primers that are
located more than 2 kb upstream of the poly(A) tail of any transcript, random
hexamers should be used for the synthesis of cDNA. We specifically chose to
amplify in all samples a collagen 1(IV) PCR product of one certain
size. We doubt whether a comparison of intensity between this product and any
longer products can be used as a test for integrity of the mRNA, because
amplification efficiencies vary between PCR products of different sizes.
Our finding that microdissection for 3 h did not diminish the glomerular
collagen 1(IV) mRNA yield corresponds with an earlier report that the
mRNA for several collagen IV chains tested remained stable for even longer
periods in dissected glomeruli kept at 4°C
(12). When working with
patient material, the time necessary for dissection of the biopsy lies within
the time period of 3 h, hence ensuring that mRNA is not degraded.
In our study, treating glomeruli with collagenase IV before
permeabilization resulted in a lower RNA extraction. This finding argues
against the benefit of applying this pretreatment for improved RNA extraction
from human glomeruli as described in an earlier report
(15). We showed there was no
significant difference in cDNA recovery between a Nonidet P-40-based- and a
Triton X-100-based permeabilization solution. However, when storing glomeruli
for 1 wk at -70°C, the addition of the Nonidet P-40-based solution gave a
better preservation of the mRNA than the Triton X-100-based solution, favoring
the application of Nonidet P-40 in this protocol. After 1 mo of storage, the
collagen 1(IV) mRNA signal had decreased by approximately 95%. This
observation refines the finding in another study that glomerular mRNA levels
for various collagen IV chains were reduced considerably after storage for 9
wk at -70°C (12). We
conclude from our studies that it is preferable to perform cDNA synthesis on
microdissected, frozen glomerular tissue within 1 wk of storage.
In previous studies, ECM mRNA quantification in relation to kidney disease has been focused mainly on glomerular and total cortical tissue. Recently, however, a study was reported in which RNA was extracted from both microdissected glomerular and interstitial tissue for the assessment of mRNA levels for ECM and ECM-regulating molecules (16). We sought to optimize the RNA isolation from microdissected interstitial tissue, and compared three RNA isolation methods with respect to their efficiency of RNA extraction. The higher RNA yield we obtained with the SGMS method, compared with the GITC/PC and LiCl/PC methods, may be explained by the fact that the SGMS method is based on the elution of bound RNA from a silica gel-based membrane. With the GITC/PC and LiCl/PC methods, phenol extraction and alcohol precipitation steps are required, which probably account for the lower RNA recovery. On the basis of these results, we recommend using the SGMS method for the isolation of RNA from small interstitial tissue specimens.
Finally, we showed that it is possible to detect collagen 1(IV) mRNA
with RT-PCR in cortical RNA isolated from nonmicrodissected, frozen kidney
biopsies that had been stored for at least 10 yr at -70°C. Microdissection
of this tissue after defrosting results in degradation of the mRNA.
In conclusion, we have described an improved method for RNA extraction from
both microdissected glomerular and interstitial tissue, and its potential for
measurement of collagen 1(IV) mRNA in both tissues.
|
Acknowledgments |
|---|
|
Footnotes |
|---|
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
2IV collagen mRNA expression by competitive
polymerase chain reaction. J Exp Med176
: 1571-1576,1992This article has been cited by other articles:
 |
|
 |
|
  M. C. Roos-van Groningen, E. M. Scholten, P. M. Lelieveld, A. T. Rowshani, H. J. Baelde, I. M. Bajema, S. Florquin, F. J. Bemelman, E. de Heer, J. W. de Fijter, et al. Molecular Comparison of Calcineurin Inhibitor-Induced Fibrogenic Responses in Protocol Renal Transplant Biopsies J. Am. Soc. Nephrol., March 1, 2006; 17(3): 881 - 888. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Eikmans, H. J. Baelde, E. de Heer, and J. A. Bruijn Messenger RNA assessment in clinical nephrology: perspectives and progress of methodology Nephrol. Dial. Transplant., December 1, 2005; 20(12): 2598 - 2601. [Full Text] [PDF]
|
|
|
|
|
|
M. Eikmans, M. C. Roos-van Groningen, Y. W.J. Sijpkens, J. Ehrchen, J. Roth, H. J. Baelde, I. M. Bajema, J. W. de Fijter, E. de Heer, and J. A. Bruijn Expression of Surfactant Protein-C, S100A8, S100A9, and B Cell Markers in Renal Allografts: Investigation of the Prognostic Value J. Am. Soc. Nephrol., December 1, 2005; 16(12): 3771 - 3786. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. Koop, M. Eikmans, H. J. Baelde, H. Kawachi, E. de Heer, L. C. Paul, and J. A. Bruijn Expression of Podocyte-Associated Molecules in Acquired Human Kidney Diseases J. Am. Soc. Nephrol., August 1, 2003; 14(8): 2063 - 2071. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Eikmans, H. J. Baelde, E. C. Hagen, L. C. Paul, P. H. C. Eilers, E. de Heer, and J. A. Bruijn Renal mRNA Levels as Prognostic Tools in Kidney Diseases J. Am. Soc. Nephrol., April 1, 2003; 14(4): 899 - 907. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 D Barbaric, L Dalla-Pozza, and J A Byrne A reliable method for total RNA extraction from frozen human bone marrow samples taken at diagnosis of acute leukaemia J. Clin. Pathol., November 1, 2002; 55(11): 865 - 867. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
|
HOME
CURRENT ISSUE
ARCHIVES
JASN Express
ONLINE SUBMISSION
AUTHOR INFO
EDITORIAL BOARD SUBSCRIBE FEEDBACK ALERTS HELP |
