Improvement of extraction and processing of RNA from renal biopsies Marian C. Roos-van Groningen, Michael Eikmans, Hans J. Baelde, Emile D.E. Heer, Jan A. Bruijn  Kidney International  Volume 65, Issue 1, Pages 97-105 (January 2004) DOI: 10.1111/j.1523-1755.2004.00366.x Copyright © 2004 International Society of Nephrology Terms and Conditions

Figure 1 RNAlater study protocol. Renal cortex was obtained from a cadaveric donor kidney and each step was applied in triplicate. RNAlater 1:8 as incubation compound was tested in the same manner as RNAlater, depicted in the two trees on the right. Abbreviations are: glom, glomerulus; T.I., tubulointerstitium; microdiss., microdissection; o/n, overnight. Kidney International 2004 65, 97-105DOI: (10.1111/j.1523-1755.2004.00366.x) Copyright © 2004 International Society of Nephrology Terms and Conditions

Figure 2 Optimization of a method for RNA extraction and effect of tissue storage on RNA yields. Three methods of extraction were compared. (▪) Trizol extraction [storage in phosphate-buffered saline (PBS)]; (▴) RNeasy extraction (storage in PBS); (○) RNeasy extraction (storage in RLT buffer); (♦) NP40 extraction (storage in NP40). Collagen α1(IV) mRNA levels were assessed with real-time polymerase chain reaction (PCR). Data represent the mean of duplicate measurements. Trizol and RNeasy spin columns for RNA extraction lead to better results than use of the NP40 method. Kidney International 2004 65, 97-105DOI: (10.1111/j.1523-1755.2004.00366.x) Copyright © 2004 International Society of Nephrology Terms and Conditions

Figure 3 Optimization of cDNA synthesis. cDNA yields were compared with different reverse transcriptase (RT) enzymes for cDNA synthesis. As a read-out system, collagen α1(IV) and transforming growth factor-β (TGF-β) mRNA were assessed with real-time polymerase chain reaction (PCR). cDNA yields were significantly higher through the use of AMV RT in comparison with Sensiscript+ and Superscript®. *P < 0.001; #P < 0.005 versus results in AMV RT. AMV RT is avian myeloblastosis virus reverse transcriptase. Kidney International 2004 65, 97-105DOI: (10.1111/j.1523-1755.2004.00366.x) Copyright © 2004 International Society of Nephrology Terms and Conditions

Figure 4 RNA integrity in whole renal cortex stored at 4°C. Comparison of the effect of phosphate-buffered saline (PBS) and RNAlater on RNA integrity in whole renal cortex stored at 4°C was assessed by RNA gel electrophoresis. Incubation of human renal cortex in RNAlater solution maintained RNA integrity over a period of 3months. The photograph shows a representative result of triplicate measurements. Kidney International 2004 65, 97-105DOI: (10.1111/j.1523-1755.2004.00366.x) Copyright © 2004 International Society of Nephrology Terms and Conditions

Figure 5 Microdissection of renal tissue in phosphate-buffered saline (PBS) and RNAlater. Renal cortex microdissected in PBS (A), RNAlater (C), or RNAlater 1:8 (E) is depicted (magnification 6.5×). Microdissection of tissues incubated in either RNAlater or RNAlater 1:8 presented problems. Glomeruli obtained through microdissection (B, D, and F) in RNAlater had lost their round-shaped appearances (D, magnification 25×) and were difficult to distinguish from surrounding tubulointerstitial debris. Kidney International 2004 65, 97-105DOI: (10.1111/j.1523-1755.2004.00366.x) Copyright © 2004 International Society of Nephrology Terms and Conditions

Figure 6 Glomerular yields after microdissection in phosphate-buffered saline (PBS), RNAlater, or RNAlater 1:8. A significantly lower number of glomeruli could be obtained from tissues dissected in RNAlater. *P < 0.005 vs. microdissection in PBS. Kidney International 2004 65, 97-105DOI: (10.1111/j.1523-1755.2004.00366.x) Copyright © 2004 International Society of Nephrology Terms and Conditions

Figure 7 RNA yields were obtained from microdissected renal biopsies. Glomerular samples (A) and tubulointerstitial samples (B) obtained through microdissection of tissue treated with phosphate-buffered saline (PBS), RNAlater, or RNAlater 1:8 were assessed using real-time PCR for collagen α1(IV). The graphs display relative mRNA levels, which have been corrected for the number of glomeruli (glomerular samples) or mg tubulointerstitial tissue (tubulointersitial samples). *P < 0.01; #P < 0.05. Kidney International 2004 65, 97-105DOI: (10.1111/j.1523-1755.2004.00366.x) Copyright © 2004 International Society of Nephrology Terms and Conditions

Figure 8 Periodic acid-Schiff (PAS) staining of the renal cortex. Histology of the renal cortex stored in either phosphate-buffered saline (PBS), RNAlater, or RNAlater 1:8 was compared through PAS stainings (magnification 40×). Glomerular morphology in RNAlater treated tissue is altered, with the glomerular capsules appearing more wrinkled. Tissue treated with RNAlater 1:8 had dispersed cytoplasma, and the cell nuclei had disappeared. Kidney International 2004 65, 97-105DOI: (10.1111/j.1523-1755.2004.00366.x) Copyright © 2004 International Society of Nephrology Terms and Conditions

Figure 9 Immunofluorescence stainings for IgA, IgG, C1Q, and C3. The left panels represent stainings on renal cortex from a patient with lupus nephritis. Stainings were performed according to standard diagnostic practice. The renal cortex from a patient with lupus nephritis, which was stored in RNAlater (right panels), displayed a dispersed staining pattern and a decreased intensity of the fluorescent signal. Kidney International 2004 65, 97-105DOI: (10.1111/j.1523-1755.2004.00366.x) Copyright © 2004 International Society of Nephrology Terms and Conditions