Selection and Validation of Reference Genes for RT-qPCR Analysis in <i>Spinacia oleracea</i> under Abiotic Stress

<div>Pairwise variation (<span class="nowrap"><svg height="8.8423pt" id="M15" style="vertical-align:-0.2064009pt" version="1.1" viewbox="-0.0498162 -8.6359 9.35121 8.8423" width="9.35121pt" xmlns="http://www.w3.org/2000/svg" xmlns:xlink="http://www.w3.org/1999/xlink"><g transform="matrix(.013,0,0,-0.013,0,0)"><path d="M697 650H468L461 623L492 619C539 613 547 605 518 546C481 471 367 264 278 116H276C239 278 197 500 186 567C180 604 185 613 226 619L252 623L260 650H24L17 623C78 617 92 613 108 533L216 -11H247C365 200 515 462 560 529C616 612 624 615 689 623L697 650Z"></path></g></svg>)</span> analysis of the candidate reference genes. The geNorm software was used to analyze the pairwise variation (<span class="nowrap"><svg height="12.689pt" id="M16" style="vertical-align:-3.40067pt" version="1.1" viewbox="-0.0498162 -9.28833 40.5735 12.689" width="40.5735pt" xmlns="http://www.w3.org/2000/svg" xmlns:xlink="http://www.w3.org/1999/xlink"><g transform="matrix(.013,0,0,-0.013,0,0)"><path d="M697 650H468L461 623L492 619C539 613 547 605 518 546C481 471 367 264 278 116H276C239 278 197 500 186 567C180 604 185 613 226 619L252 623L260 650H24L17 623C78 617 92 613 108 533L216 -11H247C365 200 515 462 560 529C616 612 624 615 689 623L697 650Z"></path></g><g transform="matrix(.0091,0,0,-0.0091,7.332,3.132)"><path d="M504 89L488 119C453 85 428 69 416 69C408 69 405 75 412 102L462 304C492 438 460 451 436 451S388 441 356 423C311 397 229 336 170 256H168L189 340C208 418 200 451 177 451C144 451 82 413 24 352L40 322C65 345 98 369 108 369C114 369 119 363 112 335L28 -3L36 -12C54 -3 83 4 112 10C125 73 138 122 153 174C205 258 328 382 376 382C395 382 399 369 384 305L330 93C312 25 323 -12 351 -12C378 -12 439 21 504 89Z"></path></g><g transform="matrix(.013,0,0,-0.013,12.507,0)"><path d="M368 703H309L44 -163H104L368 703Z"></path></g><g transform="matrix(.013,0,0,-0.013,17.868,0)"><path d="M697 650H468L461 623L492 619C539 613 547 605 518 546C481 471 367 264 278 116H276C239 278 197 500 186 567C180 604 185 613 226 619L252 623L260 650H24L17 623C78 617 92 613 108 533L216 -11H247C365 200 515 462 560 529C616 612 624 615 689 623L697 650Z"></path></g><g transform="matrix(.0091,0,0,-0.0091,25.2,3.132)"><path d="M504 89L488 119C453 85 428 69 416 69C408 69 405 75 412 102L462 304C492 438 460 451 436 451S388 441 356 423C311 397 229 336 170 256H168L189 340C208 418 200 451 177 451C144 451 82 413 24 352L40 322C65 345 98 369 108 369C114 369 119 363 112 335L28 -3L36 -12C54 -3 83 4 112 10C125 73 138 122 153 174C205 258 328 382 376 382C395 382 399 369 384 305L330 93C312 25 323 -12 351 -12C378 -12 439 21 504 89Z"></path></g><g transform="matrix(.0091,0,0,-0.0091,29.859,3.132)"><path d="M556 236V289H337V504H275V289H56V236H275V-4H337V236H556Z"></path></g><g transform="matrix(.0091,0,0,-0.0091,35.419,3.132)"><path d="M389 0V32C297 38 291 46 291 118V635C234 613 175 595 109 583V556L161 554C203 552 207 547 207 497V118C207 46 201 38 110 32V0H389Z"></path></g></svg>)</span> between the normalization factors (NF) <svg height="11.8161pt" id="M17" style="vertical-align:-3.291121pt" version="1.1" viewbox="-0.0498162 -8.52498 21.8724 11.8161" width="21.8724pt" xmlns="http://www.w3.org/2000/svg" xmlns:xlink="http://www.w3.org/1999/xlink"><g transform="matrix(.013,0,0,-0.013,0,0)"><path d="M719 650H483V622C553 619 576 606 581 560C585 532 588 487 588 394V148H585L167 650H20V622C67 618 88 610 109 585C127 562 129 557 129 484V264C129 173 126 124 123 93C118 44 92 31 35 28V0H272V28C204 32 181 44 176 95C173 124 170 172 170 264V515H172L598 -9H629V394C629 487 631 532 635 563C639 606 663 619 719 622V650Z"></path></g><g transform="matrix(.013,0,0,-0.013,9.695,0)"><path d="M493 503C489 551 484 614 483 650H43V622C120 616 128 611 128 525V126C128 40 120 34 40 28V0H312V28C221 34 213 40 213 126V307H316C407 307 412 296 424 227H453V420H424C412 355 407 346 316 346H213V584C213 613 216 616 246 616H322C398 616 419 607 436 579C449 559 455 539 464 499L493 503Z"></path></g><g transform="matrix(.0091,0,0,-0.0091,16.598,3.132)"><path d="M504 89L488 119C453 85 428 69 416 69C408 69 405 75 412 102L462 304C492 438 460 451 436 451S388 441 356 423C311 397 229 336 170 256H168L189 340C208 418 200 451 177 451C144 451 82 413 24 352L40 322C65 345 98 369 108 369C114 369 119 363 112 335L28 -3L36 -12C54 -3 83 4 112 10C125 73 138 122 153 174C205 258 328 382 376 382C395 382 399 369 384 305L330 93C312 25 323 -12 351 -12C378 -12 439 21 504 89Z"></path></g></svg> and <svg height="11.9257pt" id="M18" style="vertical-align:-3.400721pt" version="1.1" viewbox="-0.0498162 -8.52498 31.9017 11.9257" width="31.9017pt" xmlns="http://www.w3.org/2000/svg" xmlns:xlink="http://www.w3.org/1999/xlink"><g transform="matrix(.013,0,0,-0.013,0,0)"><path d="M719 650H483V622C553 619 576 606 581 560C585 532 588 487 588 394V148H585L167 650H20V622C67 618 88 610 109 585C127 562 129 557 129 484V264C129 173 126 124 123 93C118 44 92 31 35 28V0H272V28C204 32 181 44 176 95C173 124 170 172 170 264V515H172L598 -9H629V394C629 487 631 532 635 563C639 606 663 619 719 622V650Z"></path></g><g transform="matrix(.013,0,0,-0.013,9.695,0)"><path d="M493 503C489 551 484 614 483 650H43V622C120 616 128 611 128 525V126C128 40 120 34 40 28V0H312V28C221 34 213 40 213 126V307H316C407 307 412 296 424 227H453V420H424C412 355 407 346 316 346H213V584C213 613 216 616 246 616H322C398 616 419 607 436 579C449 559 455 539 464 499L493 503Z"></path></g><g transform="matrix(.0091,0,0,-0.0091,16.598,3.132)"><path d="M504 89L488 119C453 85 428 69 416 69C408 69 405 75 412 102L462 304C492 438 460 451 436 451S388 441 356 423C311 397 229 336 170 256H168L189 340C208 418 200 451 177 451C144 451 82 413 24 352L40 322C65 345 98 369 108 369C114 369 119 363 112 335L28 -3L36 -12C54 -3 83 4 112 10C125 73 138 122 153 174C205 258 328 382 376 382C395 382 399 369 384 305L330 93C312 25 323 -12 351 -12C378 -12 439 21 504 89Z"></path></g><g transform="matrix(.0091,0,0,-0.0091,21.257,3.132)"><path d="M556 236V289H337V504H275V289H56V236H275V-4H337V236H556Z"></path></g><g transform="matrix(.0091,0,0,-0.0091,26.818,3.132)"><path d="M389 0V32C297 38 291 46 291 118V635C234 613 175 595 109 583V556L161 554C203 552 207 547 207 497V118C207 46 201 38 110 32V0H389Z"></path></g></svg> in order to determine the optimal number of candidate reference genes required for RT-qPCR data normalization. If <svg height="12.689pt" id="M19" style="vertical-align:-3.40067pt" version="1.1" viewbox="-0.0498162 -9.28833 77.2621 12.689" width="77.2621pt" xmlns="http://www.w3.org/2000/svg" xmlns:xlink="http://www.w3.org/1999/xlink"><g transform="matrix(.013,0,0,-0.013,0,0)"><path d="M697 650H468L461 623L492 619C539 613 547 605 518 546C481 471 367 264 278 116H276C239 278 197 500 186 567C180 604 185 613 226 619L252 623L260 650H24L17 623C78 617 92 613 108 533L216 -11H247C365 200 515 462 560 529C616 612 624 615 689 623L697 650Z"></path></g><g transform="matrix(.0091,0,0,-0.0091,7.332,3.132)"><path d="M504 89L488 119C453 85 428 69 416 69C408 69 405 75 412 102L462 304C492 438 460 451 436 451S388 441 356 423C311 397 229 336 170 256H168L189 340C208 418 200 451 177 451C144 451 82 413 24 352L40 322C65 345 98 369 108 369C114 369 119 363 112 335L28 -3L36 -12C54 -3 83 4 112 10C125 73 138 122 153 174C205 258 328 382 376 382C395 382 399 369 384 305L330 93C312 25 323 -12 351 -12C378 -12 439 21 504 89Z"></path></g><g transform="matrix(.013,0,0,-0.013,12.507,0)"><path d="M368 703H309L44 -163H104L368 703Z"></path></g><g transform="matrix(.013,0,0,-0.013,17.868,0)"><path d="M697 650H468L461 623L492 619C539 613 547 605 518 546C481 471 367 264 278 116H276C239 278 197 500 186 567C180 604 185 613 226 619L252 623L260 650H24L17 623C78 617 92 613 108 533L216 -11H247C365 200 515 462 560 529C616 612 624 615 689 623L697 650Z"></path></g><g transform="matrix(.0091,0,0,-0.0091,25.2,3.132)"><path d="M504 89L488 119C453 85 428 69 416 69C408 69 405 75 412 102L462 304C492 438 460 451 436 451S388 441 356 423C311 397 229 336 170 256H168L189 340C208 418 200 451 177 451C144 451 82 413 24 352L40 322C65 345 98 369 108 369C114 369 119 363 112 335L28 -3L36 -12C54 -3 83 4 112 10C125 73 138 122 153 174C205 258 328 382 376 382C395 382 399 369 384 305L330 93C312 25 323 -12 351 -12C378 -12 439 21 504 89Z"></path></g><g transform="matrix(.0091,0,0,-0.0091,29.859,3.132)"><path d="M556 236V289H337V504H275V289H56V236H275V-4H337V236H556Z"></path></g><g transform="matrix(.0091,0,0,-0.0091,35.419,3.132)"><path d="M389 0V32C297 38 291 46 291 118V635C234 613 175 595 109 583V556L161 554C203 552 207 547 207 497V118C207 46 201 38 110 32V0H389Z"></path></g><g transform="matrix(.013,0,0,-0.013,44.036,0)"><path d="M512 -3V55L134 254V256L512 456V514L75 281V230L512 -3Z"></path></g><g transform="matrix(.013,0,0,-0.013,55.299,0)"><path d="M241 635C89 635 35 457 35 312C35 153 89 -12 240 -12C390 -12 443 166 443 312C443 466 390 635 241 635ZM238 602C329 602 354 454 354 312C354 172 330 22 240 22C152 22 124 173 124 313S148 602 238 602Z"></path></g><g transform="matrix(.013,0,0,-0.013,61.539,0)"><path d="M113 -12C146 -12 170 11 170 46C170 78 146 103 114 103S58 78 58 46C58 11 82 -12 113 -12Z"></path></g><g transform="matrix(.013,0,0,-0.013,64.503,0)"><path d="M384 0V27C293 34 287 42 287 114V635C232 613 172 594 109 583V559L157 557C201 555 205 550 205 499V114C205 42 199 34 109 27V0H384Z"></path></g><g transform="matrix(.013,0,0,-0.013,70.743,0)"><path d="M153 550H386L412 615L406 623H120L82 318C104 327 142 338 184 338C294 338 347 275 347 187C347 112 305 39 221 39C160 39 119 71 97 89C88 97 80 96 71 90C59 80 50 67 49 57C48 45 52 36 66 23C80 9 123 -12 169 -12C221 -11 288 15 342 59C403 109 431 165 431 225C431 308 366 395 238 395C212 395 165 379 127 364L153 550Z"></path></g></svg> (gray dotted line), the minimum value of <svg height="6.1673pt" id="M20" style="vertical-align:-0.2063904pt" version="1.1" viewbox="-0.0498162 -5.96091 6.6501 6.1673" width="6.6501pt" xmlns="http://www.w3.org/2000/svg" xmlns:xlink="http://www.w3.org/1999/xlink"><g transform="matrix(.013,0,0,-0.013,0,0)"><path d="M495 86L479 114C446 82 419 66 409 66C401 66 401 72 406 97C420 166 436 231 453 297C489 435 454 448 428 448C406 448 384 439 354 422C305 394 222 327 161 247H159L183 345C200 415 194 448 173 448C143 448 82 410 23 351L38 325C64 349 95 371 105 371C111 371 116 365 109 336L25 -4L31 -12C50 -4 77 3 107 9C119 69 132 122 145 168C197 254 321 381 370 381C387 381 393 374 378 305L329 95C309 17 320 -12 345 -12C372 -12 430 19 495 86Z"></path></g></svg> is the optimal number of genes required.</div>

BioMed Research International

fig4

Figure 4

Figure 4: Selection and Validation of Reference Genes for RT-qPCR Analysis in <i>Spinacia oleracea</i> under Abiotic Stress 

BioMed Research International

Selection and Validation of Reference Genes for RT-qPCR Analysis in Spinacia oleracea under Abiotic Stress

Figure 4