Processing renal biopsies for diagnostic mRNA quantification: improvement of RNA extraction and storage conditions.
Author(s): Eikmans M, Baelde HJ, De Heer E, Bruijn JA
Publication: J Am Soc Nephrol, 2000, Vol. 11, Page 868-73
PubMed ID: 10770964 PubMed Review Paper? No
Purpose of Paper
This paper compared mRNA yield and stability in minute quantities of renal specimens collected postmortem and subjected to an extraction delay, different RNA extraction conditions, and short- and long-term storage at -70°C of microdissected and intact renal biopsy specimens, respectively.
Conclusion of Paper
Collagen α1 (IV) mRNA levels were not altered when microdissected glomeruli were kept for 3 h at 4°C prior to RNA extraction when compared to controls extracted immediately. Permeabilization with Nonidet P-40 or Triton X-100 during RNA extraction resulted in comparable collagen α1 (IV) mRNA levels when glomeruli were processed immediately. However, storage of glomeruli at -70°C in different solutions did affect collagen α1 (IV) mRNA levels, as glomueruli stored in a solution containing Triton X-100 or rRNasin alone resulted in significantly lower levels than those stored in Nonidet P-40 for 1 or 2 weeks at -70°C. Notably, significant decreases in collagen a1(IV) mRNA were observed among glomeruli for all storage solutions after 2 and 4 weeks of storage at -70°C compared to immediately thawed and processed controls. Long-term storage of kidney biopsies at -70°C for 1, 3, 5, or 10 years did not affect collagen a1(IV) mRNA stability as determined by RT-PCR analysis.
Studies
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Study Purpose
This study investigated the effects of a delay to RNA extraction, three different methods of RNA extraction and frozen storage duration on collagen α1 (IV) mRNA levels, a low abundance gene, in microdissected glomeruli. Multiple 16-Gauge kidney biopsies were collected postmortem from three individuals, and microdissected to obtain eight batches of 10 glomeruli from each kidney. Microdissected glomeruli were exposed to RNase inhibitor (rRNasin) immediately and promptly subjected to RNA extraction or first incubated for 3 h at 4°C in phosphate buffered saline. The RNA extraction methods investigated consisted of four different permeabilization solutions: Nonidet P-40 buffer with rRNAse and Triton X-100 buffer with rRNAse with and without collegenase IV. Frozen storage experiments were performed using glomeruli that were treated with a solution containing Nonidet P-40 buffer and rRNasin, Triton X-100 and rRNasin, or rRNasin alone and immediately frozen on dry ice that were frozen and immediately defrosted or stored either for 1, 2, or 4 weeks at -70°C. RT-PCR for amplification of collagen a1(IV) mRNA was performed and cDNA levels were semi-quantitatively assessed using agarose gel electrophoresis.
Summary of Findings:
Collagen α1 (IV) mRNA levels were not altered when microdissected glomeruli were kept for 3 h at 4°C prior to RNA extraction when compared to controls extracted immediately. Permeabilization with Nonidet P-40 or Triton X-100 during RNA extraction resulted in comparable collagen α1 (IV) mRNA levels when glomeruli were processed immediately. Treatment of glomeruli with collagenase IV prior to permeabilization resulted in significantly lower levels of the collagen α1 (IV) amplicon (p<0.01). Storage of glomeruli at -70°C in different solutions did affect collagen α1 (IV) mRNA levels, as glomueruli stored in a solution containing Triton X-100 or rRNasin alone yielded significantly lower levels than those stored in Nonidet P-40 for 1 or 2 weeks at -70°C (p<0.05). Notably, significant declines in collagen a1(IV) mRNA levels were observed among glomeruli for all storage solutions after 2 weeks (P < 0.05) and 4 weeks (P < 0.01) at -70°C compared to immediately thawed and processed controls.
Biospecimens
Preservative Types
- Other Preservative
- Frozen
Diagnoses:
- Autopsy
- Normal
Platform:
Analyte Technology Platform RNA RT-PCR Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Biospecimen Acquisition Cold ischemia time 0 hrs
3 hrs
Analyte Extraction and Purification Cell/tissue permeabilization Nonidet P-40 buffer with rRNasin
Triton X-100-based solution with rRNasin
rRNasin
Collagenase IV
None
Storage Storage duration 1 wk
2 wk
4 wk
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Study Purpose
This study investigated the effects of long-term frozen storage on collagen α1 (IV) mRNA stability in kidney specimens. Five 18-Gauge kidney biopsies from patients with IgA nephropathy were stored at -70°C for 1, 3, 5, or 10 years. RNA was extracted using silica gel-based membrane spin (SGMS) technology, RT-PCR for amplification of the collagen a1(IV) mRNA was performed, and cDNA levels were semi-quantitatively assessed using agarose gel electrophoresis.
Summary of Findings:
Long-term storage of kidney biopsies at -70°C for 1, 3, 5, or 10 years did not affect collagen a1(IV) mRNA stability in total cortical tissue as determined by RT-PCR analysis.
Biospecimens
Preservative Types
- Frozen
Diagnoses:
- Other diagnoses
Platform:
Analyte Technology Platform RNA RT-PCR Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Storage Storage duration 1 yr
3 yr
5 yr
10 yr