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Purification of Amplifiable Nucleic Acid from Oil Palm and Rice Leaves and Seeds Using a Small Benchtop Automated Instrument Brad Hook1, Samantha Lewis1, Eric Ting2, Youngryul Jung3, Laurence Delaurière4, Elisa Ruiz4, Céline Ménager4, Chris Moreland1, and Trista Schagat1 1Promega Corporation, Madison, WI; 2Promega PTE LTD, Singapore; 3Promega Korea LTD, Seoul, Korea; 4PETAL, Charbonnieres, France

1. Abstract 4. Oil Palm Leaf DNA 7. Rice Kernel DNA for Genotyping

2. Maxwell® 16 Instrument for Automated Plant NA Purification

5. Oil Palm Seed DNA 8. DNA and RNA Purification from Garden and Orchard Vegetation

3. Oil Palm Leaf RNA

6. Rice Leaf DNA and RNA

9. Summary

January 2015

Plant nucleic acid (NA) purification provides a variety of unique challenges due to the broad diversity of plants, their extracellular structures, and endogenous compounds (e.g. polysaccharides, phenolics) that can co-purify with NA and inhibit downstream enzymatic assays. We evaluated a novel cellulose-based paramagnetic particle technology to purify NA from Elaeis guineensis (oil palm) and Oryza sativa (rice) plant leaf and seed lysates in an automated format using the Maxwell® 16 System. Extracted NA were evaluated for yield and amplifiability in qPCR and RT-qPCR. When compared to a competing method, the Maxwell®

16 System isolated >10x more DNA from a single oil palm leaf punch, and unlike the competing method, showed no inhibition of subsequent qPCR when 2-4 leaf punches were used for extraction. For leaf RNA and seed DNA, up to 100mg ground tissue were tested. We were able to extract amplifiable NA in both cases with no detectable co-purification of inhibitors. DNA extracted from oil palm leaves and seed was also amplifiable in endpoint PCR using SHELL gene-specific primers. DNA from a single rice kernel was sufficient for genotyping by multiplex PCR. We also tested DNA and RNA extraction from a variety of fruit and vegetable leaves including Malus domestica (apple), Vitis vinifera (grape), Citrus lemon (lemon), Solenum tuberosum (potato), and Solanum lycopersicum (tomato), and found all to yield amplifiable NA. These data support the broad utility of the novel cellulose-based technology and Maxwell® 16 System for automated plant NA purification.

• Using the Maxwell® 16 Instrument, high quality, amplifiable DNA and RNA were extracted from

• Oil palm and rice leaves • leaves from fruit, vegetable and ornamental shrubs, vines and trees.

• Amplifiable DNA was also extracted from oil palm seeds and rice kernels using a CTAB-containing buffer up front of Maxwell® 16.

• Oil palm leaf and seed DNA were compatible with PCR of the SHELL gene.

• Sufficient DNA for genotyping PCR could be extracted from single white rice kernels without grinding or bead beating.

• Brown rice kernels required grinding to maximize release of DNA.

DNA isolated from single rice kernels was amplified using the Rice Multiple-X™ PCR Kit (SolGent Co., Ltd); 5µl gDNA was added to a 25µl reaction. Six different types of rice were tested for SNP identification. Gel image of PCR products is shown.

Rice leaf RNA yields are based on quantitation using the QuantiFluor™ RNA System. GoTaq® Probe 1-Step RT-qPCR System was used to analyze purified RNA; 1µl RNA eluate was used per 20µl reaction. Changes in Ct values of samples indicate no inhibition of serial diluted eluates (data not shown).

Rice leaf DNA was extracted from 20mg leaf using the Maxwell® 16 LEV Plant DNA Kit. Yields were determined using the QuantiFluor® ONE dsDNA System. DNA quality was determined using GoTaq® Probe qPCR Master Mix and rice specific primers (4) with 1µl DNA eluate per 20µl reaction. Changes in Ct values of serially diluted samples indicate no inhibition of qPCR.

Nucleic acid was purified from plant leaves using the Maxwell® 16 LEV Plant DNA Kit (20mg input) and Maxwell® 16 LEV Plant RNA Kit (50mg input). NA was quantitated using the QuantiFluor™ dsDNA or RNA System (data not shown). A fixed volume was then assessed for amplifiability using the GoTaq® qPCR Master Mix or GoTaq® 1-Step RT-qPCR System and universal plant primers (2). * Maxwell® 16 FFS Kit (100mg input) recommended based on qPCR performance.

Leaf from DNA detected by qPCR

RNA detected by RT-qPCR

Amelanchier (amelanchier sp) √ √ Apple tree (Malus domestica) √ √ Apricot (Prunus armeniaca) √ √ Cherry tree (Prunus avium) √ √ Eggplant (Salanum melongena) √ √ Fig tree (Ficus carica) √ √ Goji (Lycium barbarum) √ √ Gooseberry (Ribes uva-crispa) √ √ Grape vine (Vitis vinifera) √ √ Kiwi (Actinidia) √ √ Lemon tree (Citrus lemon) √ √ Medlar Holland (Mespilus germanica) √ √ Medlar spain (Mespilus ermanica) √ √ Mulberry (Maus sp) √* √ Peach tree (Prunus persica) √ √ Pear tree (pyrus sp) √ √ Peony (Paeonia sp) √ √ Persimmon (Diospyros kaki) √ √ Plum (Prunus sp) √ √ Potato (Solanum tuberosum) √ √ Privet (Ligustrum sp) √ √ Quince tree (Cydonia oblonga) √ √ Raspberry (Rubus sp) √ √ Rosebush (Rosa sp) √ √ Strawberry (Fragaria ananassa) √ √ Tomato (Solanum lycopersicum) √ √ Zuchinni (Cucurbita pepo) √ √

Plant Leaf DNA: Maxwell® 16 LEV Plant DNA Kit (cat. #AS1420)

Plant Leaf RNA: Maxwell® 16 LEV Plant RNA Kit (cat. #AS1430)

Seed DNA: Maxwell® 16 FFS Kit, Custom (cat. #X9431) + CTAB Buffer (2% CTAB, 1.4M NaCl, 0.1M Tris, 10mM EDTA pH 8.0) and RNase A Solution (cat. #A7973), optional

Maxwell® 16 Instrument

OR

RNA was extracted from 20-100mg oil palm leaf using the Maxwell® 16 LEV Plant RNA Kit. Yields were determined using the QuantiFluor™ RNA System. RNA quality was assessed with GoTaq ® 1-Step RT-qPCR (GRAS primers, 1) using 1µl RNA per 20µl reaction. Difference in Ct values from serial diluted RNA indicate no inhibitors co-purified with the RNA. Gel analysis indicates RNA is intact.

Homogenize in Homogenization

Solution

+ Lysis Buffer, 10min incubation

References 1. Yeap, WC, et al. (2014) Plant Cell Tiss Organ Cult.116, 55-66. 2. Wang, J, et al. (2011) Plant Methods. 7, 39. 3. Singh, R, et al. (2013) Nature. 500, 340-344. 4. Life Technologies, Cat. #4351372.

DNA was extracted from 1-5 punches of oil palm leaf using the Maxwell® 16 LEV Plant DNA Kit. Yields were determined using the QuantiFluor™ ONE dsDNA System. GoTaq ® qPCR analysis of purified DNA (universal plant primers, 2) using 1µl DNA per 25µl reaction is shown. Difference in Ct values with serial diluted DNA indicate no inhibitors co-purified with the DNA. Gel analysis shows high molecular weight DNA was isolated.

SHELL gene (3) was amplified using GoTaq® Master Mix and DNA isolated from 1-5 leaf punches. 2µl DNA eluate was added per 25µl reaction. 1.0% agarose gel analysis of each amplification is shown.

Lysis Buffer + RNase A

DNA was extracted from 20-100mg oil palm seed using the Maxwell® 16 FFS Kit. Yields were determined using the QuantiFluor™ ONE dsDNA System. DNA quality was assessed by GoTaq ® qPCR (with universal plant primers, 2) using 1µl DNA per 25µl reaction. Difference in Ct values with serial diluted DNA indicate no inhibitors co-purified with the DNA. SHELL gene (3) was amplified using GoTaq® Master Mix and 2µl DNA per 25µl reaction. 1.0% agarose gel analysis of each amplification is shown.

CTAB + Proteinase K +

RNase A

Rice Input [DNA] (ng/µl)

Medium Grain White

Kernel 35±10.4 Ground (100mg) 17.5±2.1

Brown Kernel 2.1±0.7

Ground (100mg) 13±2.8

Basmati White Kernel 28.3±5.1 Brown Ground (100mg) 23.7±11.5

DNA was extracted from 5 whole kernels or 100mg ground kernel using the Maxwell® 16 FFS Kit. Yields were determined using the QuantiFluor™ ONE dsDNA System. DNA quality was assessed using GoTaq ® qPCR and rice-specific primers (4).